The cytochrome P450 I gene family of microsomal hemoproteins and their role in the metabolic activation of chemicals.

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DRUG METABOLISM REVIEWS, 22(1), 1-85 (1990)

THE CYTOCHROME P450 I GENE FAMILY OF MICROSOMAL HEMOPROTEINS AND THEIR ROLE IN THE METABOLIC ACTIVATION OF CHEMICALS* C. IOA"IDESt and D. V. PARKE Department of Biochemistry University of Surrey Guildford, Surrey, GU2 5XH, U.K.

I. 11. 111.

IV.

...................................

3

CYTOCHROME P-450GENE FAMILIES AND NOMENCLATURE ..................................

5

SIMILARITIES BETWEEN CYTOCHROMES P-448 AND OTHER CYTOCHROME P-450FAMILIES. . . . . . . . . . . . . . A. Relationship to Cytochrome P-450I1 A . .............. B. Constitutive and Inducible Levels ....................

6 6 6

DIFFERENCES BETWEEN CYTOCHROMES P-448AND OTHER CYTOCHROME P-450FAMILIES. . . . . . . . . . . . . . A. Primary Structure ................................. B. mRNA Studies ...................................

9 9 12

INTRODUCTION

*This paper was refereed by James R. Gillette, Ph.D., Chief, Laboratory of Chemical Pharmacology, National Heart, Lung, and Blood Institute, NIH, Bethesda, Maryland 20892. ?To whom correspondence should be sent. 1 Copyright 8 1990 by Marcel Dekker, Inc.

IOANNIDES AND PAR=


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C. cDNA Nucleotide Sequences ........................ D. Response to Inducing Agents ........................ E. Immunological Studies ............................. F. Substrate Binding Sites ............................. G. Substrate Specificities.............................. H. Metabolic Activation and Detoxication . . . . . . . . . . . . . . . . I. Metabolism of Endogenous Substrates ................ J . Regulatory Control ................................ K . Hemoprotein Turnover ............................. L. Hepatocellular and Pulmonary Distribution.............

13 14 14 15 19 20 22 23 23 25

V . METABOLIC ACTIVATION OF CHEMICALS .......... A. Polycyclic Aromatic Hydrocarbons . . . . . . . . . . . . . . . . . . . B. Polyhalogenated Biphenyls ......................... C. Aromatic Amines ................................. D. Aromatic h i d e s ................................. E. Azo Compounds .................................. F. Aflatoxin B, ...................................... G. Drugs and Other Chemicals......................... H. Flavonoids....................................... I. Nitrosamines ..................................... J . TCDD ..........................................

25 25 28 29 30 31 31 32 33 33 34

VI .

MECHANISM OF ENZYME INDUCTION: THE Ah LOCUS ............................................ A. The Ah Receptor ................................. B. The Ah Receptor and Immunotoxicity . . . . . . . . . . . . . . . . C. Ah Receptor Binding and Cytochrome P-448 Induction .. D. Dimensions of the Ah Receptor...................... E. Regulation of Cytochrome P-450d.................... F. Species Differences ................................ G. Further Aspects of the Ah Locus.....................

34 34 36 37 37 39 39 40

VII. CYTOCHROMES P-448 AND CHEMICAL TOXICITY. . . . A. Paracetamol ...................................... B. Polycyclic Aromatic Hydrocarbons ................... C. Aromatic Amines and Amides ....................... D. Azobenzenes ..................................... E. Polyhalogenated Biphenyls.......................... F. Multidrug Resistance...............................

41 42 42 44 44 45 45

VIII.

FACTORS MODULATING CYTOCHROME P-448 ACTIVITY ......................................... A. Tissues..........................................

46 46

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CYTOCHROME P450 I GENE FAMILY

B. C. D. E. F.

Sex ............................................. Species Differences. ............................... Strain Differences. ................................ Age.. ........................................... Nutrition.. .......................................

51 52 54 55 56

....................

57

IX.

HUMAN CYTOCHROMES P-448..

X.

COMPETITION BEWEEN CYTOCHROMES P-448 AND PBCYTOCHROMES P-450 ..........................

60

CONCLUSIONS. ....................................

61

References ..........................................

62

XI.

I. INTRODUCTION No enzyme system has attracted the attention of researchers more than the cytochrome P-450-dependent mixed-functionoxidases, an enzyme system that displays unprecedentedly broad substrate specificity, catalyzing the oxidation and reduction of a plethora of substrates, both endogenous and exogenous, of widely differing chemical structures and physicochemical properties. Although the role of the cytochromes P-450 in the metabolism of endogenous substrates is still not fully elucidated, they are known to play a critical role in the synthesis and catabolism of fatty acids, cholesterol, steroid hormones, and eicosanoids. The cytochrome P-450 hemoproteins, the terminal oxygenases of the mixed-function oxidase system, probably occur in all living organisms and have been demonstrated in bacteria, yeasts, plants, insects, fish, and mammalia. In the cell, the mixed-function oxvidases are localized in the mitochondria, the nuclear envelope, and the endoplasmic reticulum (microsomes). The mitochondrial cytochromes P-450 participate in the biosynthesis of the steroid hormones and bile acids, as well as in the metabolism of vitamin D,; they also catalyze the metabolism of certain xenobiotics, and have been shown to effect the N-demethylations of ethylmorphine and benzphetamine and to metabolically activate chemical carcinogens to their toxic intermediates [1-31. The microsomal cytochromes P-450, in addition to their function as steroid oxygenases, are responsible for the oxygenation of lipophilic xenobiotics, largely resulting in increased polarity and loss of biological activity. The physiological significance of the nuclear cytochromes P-450, which in many respects are similar to the microsomal hemoproteins, has not yet been elucidated, but they are also involved in the activation of chemicals such as the polycyclic aromatic hydrocarbons (PAH) and heterocyclic aromatic amines [4, 51. The concentrations of the cytochromes P-450 from all three different


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IOANNIDES AND PARKE

subcellular organelles are increased following treatment of animals with chemical inducing agents, and the newly synthesized proteins are related to the constitutive enzymes, at least immunologically. The hepatic microsomal and nuclear cytochromes P-450 are antigenically similar [3], and rat 3-methylcholanthrene (MC)-induced nuclear and microsomal cytochromes are also immunologically similar [6]. Nuclear cytochromes P-450 are induced by many known inducers of the microsomal cytochromes, such as pregnenolone 16a-carbonitrile (PCN), phenobarbitone (PB), (3-naphthoflavone (P-NF), and the chlorinated biphenyl mixture Aroclor 1254 [7]. However, the mitochondrial mixed-function oxidases differ from the microsomal and nuclear enzynies in the electron donor system, the mitochondrial forms being able to operate with adrenodoxin, ferredoxin, or the microsomal cytochrome P-450 reductase, while the microsomal and nuclear forms function only in the presence of the reductase [3]. The present review concerns the extensively studied cytochrome P-450 I gene family of the microsomal system. The wide substrate specificity of the microsomal mixed-function oxidases is due largely to the multiplicity of the constitutive and inducible cytochromes P-450, which display different, but often overlapping, substrate specificity. As the affinity of the hepatic microsomal cytochromes P-450 is much higher for the steroid hormones than for xenobiotics, steroids are generally considered to be the major endogenous substrates (8, 91. Indeed, a number of steroidoxygenating cytochrome P-450 proteins showing strict regio- and stereospecificity, and sex specificity [ 10-121 have been isolated, indicating that many other forms are likely to be present to catalyze steroid oxygenations at other positions. It might be expected that these steroid hydroxylases would not accept xenobiotics as substrates, so as to eliminate interactions between physiological substrates and synthetic chemicals. However, recent studies have shown [ l l ] that a constitutive form of cytochrome P-450 isolated from male mice and highly active in the 16a-hydroxylation of testosterone, also demethylates benzphetamine, although its rate of oxygenation of this drug was low. Similarly, a male-specific constitutive isoenzyme, isolated from the adult rat and efficient in the 2a- and 16a-hydroxylation of steroids [12], and a female-specific steroid 15fLhydroxylase [131, also exhibited high catalytic activities toward a number of xenobiotics. Cytochrome P-450 proteins can therefore be involved simultaneously in the metabolism of endogenous substrates and of structurally unrelated xenobiotics. The mixed-function-oxidase-catalyzedmetabolism of xenobiotics is primarily concerned with detoxication involving the formation of more polar, readily excretable metabolites. Paradoxically, however, the same liver microsomal mixed-function oxidase system can affect the formation of more toxic, reactive intermediates, a process known as “metabolic activation” or “bioactivation.” A likely explanation of this paradox is that the activation process may be



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attributed entirely to one or more specific gene families of the cytochrome P-450 proteins, while other gene families catalyze oxygenations which lead to detoxication [14, 151. The amount of a reactive intermediate formed is, thus, the result of these two competing processes-that is, activation and detoxication-catalyzed by different gene families of the hemoprotein, so that, not only the cellular levels but also the cytochrome P-450 isoenzyme population, will determine whether a chemical will be activated or deactivated and subsequently eliminated.

11.

CYTOCHROME P-450 GENE FAMILIES AND NOMENCLATURE

One of the earliest and most important observations was that mixed-function oxidase activity could be increased by the administration to animals of a variety of foreign chemicals which elicited the synthesis of new enzymic protein, the pattern and extent of induction being dependent on the nature of the inducing agent [ 161. Following the successful solubilization and purification of the mixed-function oxidase system, it became clear that these agents selectively induced specific isoenzymes. Cytochrome P-450 isoenzymes involved in the biosynthesis of endogenous compounds such as steroids were generally considered to be refractive to induction by foreign chemicals,but PB was shown to induce a rat liver mitochondrial C,, steroid hydroxylase [ 171, and both MC and PB induce mitochondria1cytochrome P-450 proteins. Of the many distinct gene families of cytochromes P-450 that have now been recognized [181, the two most extensively studied are those whose prototype inducing agents are PB and PAH, respectively. These two families have been solubilized, isolated, and purified from a number of animal species, by many different laboratories, and the only obstacle in comparing the various preparations is the grossly inadequate and often confusing nomenclature that is so often used. In the present review the PB-inducible forms are referred to as PB-cytochromes P-450 (P-450b and P-450e for the rat in the Levin nomenclature), and the PAH-inducible forms are referred to in their original terminology as cytochromes P-448 (P-45Oc and P-450d for the rate in the Levin nomenclature). The Levin nomenclature is also used to describe other cytochrome P-450 proteins. Although we appreciate that this terminology may be considered imprecise and anachronistic, it is adopted in the present review for the sake of simplicity. It has now been established that rat P-45Oc and P-450d correspond to rabbit forms LM, and LM, and to mouse P,-450 and P,-450, respectively [ 191. In the newly recommended nomenclature [20], the cytochromes P-448 constitute the P450 I gene family; this comprises only one subfamily, consisting of the two isoenzymes P450 I A1 and P450 I A2,which


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IOANNIDES AND P A R E

correspond to rat proteins P-45Oc and P-450d, respectively. The PBcytochromes P-450 form part of the P450 I1 gene family and make up the subfamily P450 IIB, which comprises the two isoenzymes P450 I1 B1 and P450 B2, corresponding to rat proteins P-450b and P-450e, respectively (see Table 1).

111. SIMILARITIES BETWEEN CYTOCHROMES P-448 AND

OTHER CYTOCHROME P-450 FAMILIES

A. Relationship to Cytochrome P450 I1 A Although the cytochromes P-448 (P-45Oc and P-450d) and the P R cytochromes P-450 (P-450b and P-450e) are dramatically induced by PAH and PB, respectively, they are only minor constitutive enzymes of uninduced rat liver. In contrast, cytochrome P-450a (P450 I1 A), which catalyzes the 7 ahydroxylation of steroid horomones, is relatively resistant to induction, but is a significant constitutive enzyme, comprising 5% to 10% of total rat liver cytochrome P-450. The largest increase in P-450a results from administration of MC or certain halogenated biphenyls, which also induce the cytochromes P-448 [21], thus indicating similarity between the induction of the P450 I and P450 I1 A families of cytochromes. However, cytochrome P450 I1 A is also induced by PB [22]. That MC is the more potent inducer of P450 I1 A is consistent with the observation that the 7a-hydroxylation of testosterone is more effectively induced by MC than by PB [23]. The polyhalogenated biphenyl (PCB) 3,4,5, 3’,4’,5’-hexachlorobiphenyl(HCB) induces P-450a in male rat liver more slowly (7 days) and to a lesser extent (2- to 3-fold) than it induces P-45Oc (> 200-fold, in 3 to 5 days), and although HCB induces P-45Oc dramatically in rat lung, kidney, and prostate, neither P-450a nor steroid 7a-hydroxylase activity was present in any of these extrahepatic tissues, either constitutively or after HCB treatment [24]. Cytochrome P-450a exhibits high regioselectivity, catalyzing the 7ahydroxylations of androstenedione, progesterone, and testosterone, and to a much lesser extent, the 6a-hydroxylation [25-281; and its induction is both age and sex dependent [29].

B. Constitutive and Inducible Levels Both the Phytochromes P-450 and cytochromes P-448 are found at very low levels in the untreated animal but are markedly induced by the appropriate

P450 IV

P450 I1 E P450 I11

P450 I1 A P450 I1 B(PB-P-450)

P450 I (P-448)

Family or subfamily P-45oc P-45od P-450a P-450b P-45oe P-45oj P-45op

A1 A2 A1 B1 B2 El A1 A2 A1 P-452

Rat proteins

Isoenzymes

LM,,

-

-

-

LM,

LM,

Rabbit proteins

-

-

PI-450 P3-450

Mouse proteins

Clofibrate

Ethanol and isoniazid PCN and clotrimazole

PAH Isosafrole PB and MC PB

Characteristic inducing agent

Inducible Families of Cytochrome P-450 Involved in the Metabolism of Chemicals

TABLE 1


-

-

z5

f

c (

R 0

cd

30 15

8


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IOANNIDES AND PARKE

inducing agents. They each comprise less than 5% of the constitutive cytochromes P-450-that is, the cytochrome population of the untreated rat [30, 311-and, together with cytochrome P-450a, they account for less than 25% of the constitutive cytochromes P-450 of rat liver [21]. They may be markedly induced by as much as 70-fold [29, 32,331, and when induced may comprise as much as 80% of the total rat liver microsomal cytochrorne P-450 content. However, two other cytochromes P-450 families, namely, the PCN-inducible cytochrome P-45Op and the alcohol-inducible P-450j, are present in substantial amounts in the noninduced animal [34, 351. Induction of one group of cytochrome P-450 proteins may suppress the already low levels of the other. Treatment of animals with MC and other PAH decreased the levels of P-450b and other PB-specific forms in untreated animals, and the higher levels encountered in PB-treated animals [36-391. Furthermore, exposure of rats to MC or HCB decreased the levels of the constitutive male-specific rat liver P-450h (P450 I1 C2) [40]. Similarly, coadministration of PB with the PCB mixture Aroclor 1260 decreased the amount of LM, in rabbit liver microsomes [41]. Induction of both PB-cytochromes P-450 and cytochromes P-448 is accompanied by the de novo synthesis of several other cytochrome P-450 proteins. PB induces cytochromes P-450b and P-450e, and in addition, also induces cytochrome P-450a, cytochrome P-45Op (P450111 A l b w h i c h is also induced by the synthetic steroid PCN [34], some imidazole-containing antifungal agents [42], and more effectively by macrolide antibiotics [43],-and a fifth isoenzyme which is also present in significant amounts in the untreated animal [25,44]. These findings are compatible with the observations that PB induces more than one mRNA coding for the hernoproteins [45,46]. It is likely that the synthesis of the number of hemoproteins affected by PB induction is greater than generally believed, as in recent studies, preparations considered to be homogeneous on the basis of immunoprecipitation and limited NH,-terminal sequence analysis, were found to comprise three distinct proteins [47]; the use of monoclonal antibodies will no doubt enable the identification of such closely related proteins. Similarly, five antigenically distinct forms, exhibiting different catalytic activities toward a number of substrates, have been purified from the liver of rats treated with p-NF [48]. These have not been extensively studied or compared with those cytochromes increased by other classes of inducers, to establish the selectivity of induction by P-NF and other inducers of this class. However, in addition to the two major forms P-45Oc and P-450d [32], a third isozyme which also has a CO-reduced cytochrome absorption maximum of 448 nm has been isolated from the liver of MC-treated rats [49]. Generally, it appears that there are more cytochrorne P-450 proteins induced by the PB class of inducing agents than there are cytochromes P-448 induced by the MC class of inducers.



9

Another inducible cytochrome P-450 with a significant role in the metabolism of chemicals is the alcohol-inducible P-45Oj (P450 I1 E) in the rat and its orthologous LM,, in the rabbit [50, 511; this cytochrome P-450 protein is inducible by small molecules such as ethanol, acetone, benzene, isoniazid, and imidazole. A further additional family (P450 IV or cytochrome P-452) is induced by lipophilic esters and ethers, such as diethylhexyl phthalate and clofibrate [52-541.

IV. DIFFERENCES BETWEEN CYTOCHROMES P-448 AND OTHER CYTOCHROME P-450 FAMILIES Despite the similarities outlined in the preceding section, there is overwhelming evidence, based on a number of experimental criteria, that demonstrates unequivocally that cytochromes P-448 differ from the PBinducible (see Table 2) and other families of cytochromes, and although evolved from a common ancestor, that they are members of distinctly different gene families.

A. Primary Structure The primary amino acid sequence of cytochromes P-448 and PB-cytochromes P-450 were the first to be deduced. A high homology of 68% has been observed between rat cytochromes P-45Oc and P-450d [55-571, although these exhibit different NH,- and COOH-terminal amino acid sequences [58]. However, the homology between cytochrome P-45Oc and the PB-inducible cytochromes P-450b and P-450e is only 29% [57, 591. Similarly, the MC-inducible murine cytochromes P,-450 and P,-450 share a protein homology of 73% and exhibit a 93% homology with the orthologous rat proteins, respectively, but homology with the PB-inducible rat cytochrome P-450e and rabbit LM, is only 15% [ 191. The PB-inducible cytochromes P-450b and P-450e share an even higher homology of more than 97%, differing only in 13 amino acids in a part of the molecule comprising 491 amino acids [59, 601 and possessing identical NH,-terminal sequences for the first 302 amino acids. Cytochrome P-45Oj (P450 I1 El), which is induced in rat by alcohol, has virtually no similarity with cytochromes P-448 but shares 48% homology with the two rat PB-inducible proteins [61]. The amino acid sequence of LM,,, the cytochrome P-450 (P450 I1 El) induced in rabbit by alcohol, has recently been determined and was found to share 48% homology with LM, but less than 30% with either LM, or LM, [62]. Another isozyme of cytochrome P-450, namely cytochrome P-452 (P450 IV), which catalyzes the o-hydroxylation of lauric acid [52], has less than 35% amino acid similarity with cytochromes P-448 and

IOANNIDES AND PARKE

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TABLE 2 Differences Between PB-Cytochrornes P-450 and Cytochromes P-448 Property Number of isozymes

PB-Cytochrornes P-450 (P450 I1 B) At least 5

Enzymic characteristics 450 nm CO absorption maximum High affinity Interaction with reductase Apoprotein turnover Long half-life in untreated animals Antigenic properties Polyclonal antibodies

Monoclonal antibodies Structure

Substrate binding site Substrate specificity

Molecular dimensions of substrates Regioselectivity

Cross-reactivity among them but no interaction with cytochromes P-448 No interaction with cytochromes P-448 High amino acid and nucleotide homology among them but not with cytochromes P-448

Cytochromes P-448 (P450 I) At least 3 448 nm Low affinity Short half-life in untreated animals Cross-reactivity among them but no interaction with PB-cytochromes P-450 No interaction with Pkytochromes P-450 High amino acid and nucleotide homology among them but not with Pkytochromes P-450

Broad substrate specificity; can accept globular and planar molecules Small area/depth ratio with large depth

Narrow substrate specificity; accepts only planar molecules Large area/depth ratio with small depth

Cannot oxygenate sterically hindered positions

Oxygenates sterically hindered positions


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TABLE 2 (Continued) Property Most specific substrate Most specific inhibitor Tissue Distribution Liver Lung Kidney

PB-Cytochromes P-450 (P450 I1 B)

Cytochromes P-448 (P450 I)

7-Pentox y resoru fin

7-Ethoxyresorufin

Metyrapone

9-Hy drox y ellipticine

Present at low levels; highly inducible Present at high levels; not inducible Present at low levels; not inducible Not detectable; not inducible Not detectable; not inducible Present in rabbit aorta; present in rabbit bladder mucosa but not inducible; present in rabbit nasal mucosa but not inducible

Present at low levels; highly inducible Present at low levels; inducible Detectable; inducible

Sex differences

Higher in the male

Detectable; highly inducible Detectable; highly inducible Present and inducible in the rabbit aorta; present in rabbit bladder mucosa, not inducible; present in induced rat ventral prostate; present in rabbit nasal mucosa; present in induced rat mammary microsomes Higher in the female

Perinatal development

Low in the neonate; increases with age until adulthood

Predominates in the neonate; decreases with age

Phenobarbitone

3-Methylcholanthrene

No receptor identified

A cytosolic Ah receptor has been identified

GI tract

Placenta Other tissues

Gene regulation Typical specific inducing agent Mechanism

(Continued)

IOANNIDES AND PARKE

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TABLE 2 (Continued) PB-Cytochromes P-450 (P450 I1 B)

Cytochromes P-448 (P450 I)

Regulatory control

Coinduced by many chemicals and under coordinate control

Strain difference

Inducible in both C57BL and DBA mice Inducible in both rats and hamsters

Coinduced by many chemicals but not under coordinate control Inducible only in C57BL mice

Property

Species differences

Perinatal development Endogenous substrates Testosterone

Prostaglandins

More inducible in the neonate Hydroxylates at 16a-, 16p-, and 17-positions Hydroxylates primarily at the o-1-position and to a lesser extent at the o-position; requires cytochrome b,

Poorly inducible in hamsters; inducible in rats More inducible in the adult Hydroxylates only at the 6P-position Hydroxylates at w-1 and 0 - 2 positions; no requirement for cytochrome b,

PB-cytochrome P-450 [63, 641. Finally, the PCN-inducible P-45Op shares a homology of around 30% with the rat and murine cytochrome P-448 proteins and similarly with the PB-inducible rat P-450e and rabbit LM, [65]. All cytochrome P-450 proteins share a common sequence at two conserved regions, and a cysteine molecule within these regions is believed to be the site for the binding of the heme to the protein [19].

B. mRNA Studies Induction of the different cytochrome P-450 proteins involves increased synthesis of specific mRNAs coding for the various cytochrome P-450 isoenzymes (66-681 and in most cases the different P-450s do not appear to arise



13

from posttranslational modifications [46]. Even closely related isoenzymes induced by the same agents are separate gene products [45, 69, 701, although cytochrome P-448 peptides may be further processed by proteolytic cleavage [711. In the rat, MC induces three mRNAs coding for cytochrome P-450 peptides, together with two other mRNAs which do not code for cytochrome P-450 proteins, indicating that this inducing agent also affects non-cytochrome P-450 genes [45]. The mRNAs that code for cytochrome P-45Oc and P-450d were increased by cytochrome P-448-inducing agents but not by PB [66]. These two mRNAs share homologous regions but also have regions that are unique; they also have different sizes and differ from each other 1721 more than do two PB-inducible mRNAs which are of similar size and differ only in a few bases [60,73,74]. These observations are compatible with the differences seen in the primary structure of these cytochromes. Mouse liver RNAs encoding for cytochromes P,-450 and P,-450 have a highly homologous region of several hundred base pairs [75]. In the rabbit, three genes are associated with the PB-induced group of cytochrome P-450 proteins; the first two mRNAs show 90% homology, and 72% homology was seen between these and a third mRNA [76].

C. cDNA Nucleotide Sequences Genomic blot analysis of DNA indicated that the MC-inducible cytochrome P-448 proteins are smaller than the PB-inducible isoenzymes [46, 77, 781. The complete cDNA sequences of the MC-inducible murine cytochromes P,-450 and P,-450 have been determined and shown to share a 68% homology [59]; these two genes are located on the same chromosome [79], display remarkable similarity in the intron-exon patterns [go], and appear to be regulated by the same cytosolic receptor protein [81], indicating that these two genes belong to the same family. Moreover, a comparison of the cDNA sequences showed 86% homology between rat P-45Oc and mouse P,-450, and 88% between rat P-450d and mouse P,-450 [19]. Furthermore, a 70% nucleotide sequence homology exists between rat P-45Oc and P-450d and mouse P,-450 [78]. The homology between P-45Oc and P-450d is particularly high in the regions coding for the carboxy-terminalamino acids [78]. In contrast, the nucleotide sequences of the murine cytochromes P,-450 and P,-450 exhibited only 30% homology with the cDNAs coding for the PB-inducible rat P-450e and rabbit LM,, demonstrating that the latter belong to a different gene family [19,56,82]. Similarly, the gene structure sequence of rat P-45Oc differed greatly from that of P-450e [83], but the latter was similar to that of P-450b [84]. All these observations indicate that the cytochrome P-448 proteins constitute a gene family markedly different from that of the PB-inducible cytochrome P-450 proteins.


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IOANNIDES AND PARKE

Finally, the cDNA sequence of cytochrome P-452 (P450 IV) shared less than 35% homology with cytochromes P-448 and Phytochromes P-450 [63, 641, and P-45Oj (P450 I1 El) had only a 50% sequence homology with P-450e (P450 I1 B2) (611, while P-45Op (P450 111A l ) shared a nucleotide sequence similarity of nearly 40% with rat and mouse cytochromes P-448 and P-450e [65].

D. Response to Inducing Agents Cytochromes P-488 and PB-cytochromes P-450 are selectively induced by different chemicals. Cytochromes P-488 are induced by planar molecules such (TCDD) and PAH [ 14,32,85], planar as 2,3,7,8-tetrachlorodibenzo-p-dioxin PCB [31,21,86], ellipticines [87,88], aminoazobenzenes [89,90], polychlorinated dibenzofurans [91], and to a lesser extent by aromatic amines [ 14,92-941, heterocyclic amines [95, 961, anthraquinones [97], and drugs like dantrolene [98]. In contrast, PB-cytochrome P-450 activity is enhanced by a variety of other chemicals, in addition to PB, including DDT [99], trans-stilbene oxide [32, 1001,y-chlordane, and SKF-525A [32], xylene [loll, and some nonplanar PCB (21). Finally, methylenedioxyphenyl compounds, such as safrole and isosafrole [32, 1021, 2-acetylaminofluorene (2-AAF) [ 103, 1041, numerous PCB [21], hexachlorobenzene [ 1051,and the drugs chlorpromazineand phenothiazine [32] act as mixed-type inducers, elevating the activities of enzymes associated with both families of cytochromes. Frequently, quantification of the various cytochrome P-450 isozymes is achieved by immunological techniques, and an erroneous assumption is made that immunorelated proteins induced by the various agents are identical. The cytochromes P-450~ isolated from livers of rats induced with MC or p-NF, had the same subunit molecular weights, electrophoresis patterns following proteolytic digestion, CO maxima, and immunological characteristics[ 1061, but their catalytic activities toward benzo(a)pyrene, although similar were not identical, indicating that although these two cytochromes P-45Oc may be structurally very similar they are distinctly different proteins having different substrate specificities.

E. Immunological Studies Polyclonal antibodies prepared against the purified hemoproteins were the first tools to distinguish the cytochrome P-450 proteins of different gene families. Such antibody preparations, however, do not generally distinguish between cytochrome P-450 proteins of the same gene family because of their high structural homology. The two major forms of cytochromes P-448 isolated



15

from the liver microsomes of rats treated with MC each cross-reacted with antibodies against the other [107]. Five of the six cysteine-containing tryptic peptides from P-450d had significant sequence homology to cysteinecontainingpeptides in P-45Oc [ 1081. Despite their antigenic similarity, these two forms of cytochromes P-448 differ in their substrate specificity, molecular weight, chromatographicproperties, and peptide maps [109]. In contrast to the two forms of cytochrome P-448, the two major PB-inducible cytochromes, P-45Ob and P-450e, are immunochemically similar and catalyze the metabolism of the same substrates, although the electrophoreticprofiles following digestion were similar but not identical, indicating minor structural differences. The preparation of a series of monoclonal antibodies to the PB-inducible cytochromes P-450 and cytochromes P-448 not only allowed the identification of different proteins within one gene family, but also further highlighted the differences between these two gene families. Nine monoclonal antibodies against cytochrome P-45Oc were prepared, three of which cross-reacted strongly with cytochrome P-450d, showing the presence of common epitopes; however, none of these antibodies cross-reacted with cytochromes P-450b or P-450e, or with any other cytochrome P-450 proteins (apart from P-45Oc and P-450d) in Western blots of hepatic microsomes from untreated or MC-treated rats [1101. Similarly, 12 monoclonal antibodies were prepared against cytochrome P-450b, of which 10 also recognized cytochrome P-450e; however, none of these cross-reacted with the cytochromes P-448 [ 1111. Polyclonal and monoclonal antibodies to P-45Oj (P450 I1 El) did not cross-react with rat PB-inducible proteins [65].

F. Substrate Binding Sites The early observation that inducing agents like MC differed from drugs such as PB in the nature of the enzymic activities they stimulated, first indicated that the cytochromes P-450 induced may have distinctly different substrate binding sites. Subsequent spectral studies revealed that pretreatment with PB increased the magnitude of the type I spectral interaction between hepatic microsomes and substrates such as hexobarbital, benzphetamine, or perhydrofluorene;while, in contrast, pretreatment with MC or other cytochrome-P-448-inducing agents either decreased or completely abolished the type I spectral interaction [112, 1131. Similar observations were made when purified rabbit proteins LM2(P450 I1 B1) and LM,(P450 IA2) were used instead of the crude microsomes . The PB-cytochromes P-450 appear to possess one substrate binding site of broad specificity which can accommodate a diversity of substrates while, in contrast, cytochromes P-448 possess a substrate binding site which exhibits narrow specificity [113]. This concept is supported by the work of Dus [114], who demonstrated homology between several different hemopeptides of PB-


16


cytochromes P-450, which comprised the heme moiety and the substrate binding site, but very little homology between these and a cytochrome P-448 hemopeptide. In order to define the molecular dimensions of the Pkytochrome P-450 and cytochrome P-448 binding sites, computergraphic techniques were employed to establish the preferred molecular geometries of specific substrates, specific inducers, and specific inhibitors of these two families of the cytochrome. Chemicals which interact specifically with cytochromes P-448 are essentially rigid, planar molecules characterized by a small depth and a large area/depth ratio [ 115,1161. Specific substrates such as 7-ethoxyresorufin, specific inducers such as MC, and specific inhibitors of cytochrome P-448 activity, such as 9-hydroxyellipticine [ 117,1181 and anthraflavic acid [ 1191, are all rigid, planar molecules (see Table 3 and Fig. 1). In contrast, compounds which elicit no interaction with cytochromes P-448, such as PB and DDT, are nonplanar, bulky molecules characterized by a small area/depth ratio and greater flexibility in molecular conformation (see Table 3 and Fig. 1). The importance of planarity in the interaction of chemicals with cytochromes P-448 is further supported by extensive studies using PCB as probes. PCB which exhibit maximum coplanar conformation by being substituted in the meta and para positions, preferentially induce cytochromes P-45Oc and P-450d. However, when planarity is decreased by introducing one chlorine atom into an ortho-position, the mono-ortho coplanar analogues exhibit mixed-type induction, increasing the levels of the PB-inducible forms of' cytochrome P-450, in addition to cytochromes P-4SOc and P-450d, which are induced to a lesser extent than before [86]. Similarly, many of the mono-ortho analogues of the coplanar polybrominated biphenyls are mixed-type inducers [37]. When two other chlorosubstituents are introduced, this results in marked loss of coplanarity and poor cytochrome P-448 inducibility [86]. Excellent correlations have been shown between the molecular dimensions of a series of polychlorinated biphenyls and the extent of induction of cytochrome P-448 in rat hepatoma cells [120]. Similar correlations have been observed between molecular planarity and the hydroxylation of dichlorobiphenyls catalyzed by the two families of the cytochrome. When the ortho positions of dichlorobiphenyl were substituted, overall rates of hydroxylation were higher with cytochrome P-450b but, in contrast, cytochrome P-45Oc readily catalyzed the hydroxylation of dichlorobiphenyls with no ortho chlorosubstituents [121]. In a structure-activity study involving 40 azo compounds, induction of cytochrome P-448 activity was achieved only by analogues that could assume coplanar structures, with three fused 6-membered rings through intramolecular hydrogen bonding [ 1221; these compounds failed to induce Pkytochromes P-450 enzyme activities. Azo compounds which induce cytochromes P-448 have molecular areas in the range of 9 k 140 A' [122], which is similar to that

15.9 13.6 15.1 14.6 14.6 13.5 14.4 13.6 11.6 12.3 14.9 13.6 10.1

Length(A)

Source. Data from Refs. 115, 116, and 120.

Dibenzo(a,h)anthracene Benzo(a)p y rene Dimethylaminoazobenzene 7-Ethoxyresorufin 3-Methylcholanthrene 9-Hy droxy ellipticine 2-Acetamidofluorene Benzo(a)pyrene-7,8-diol Paracetamol Aflatoxin SKF-525 A DDT Phenobarbitone

Molecule

(A)

9.3 9.0 6.6 9.1 8.6 9.1 7.3 9.0 7.3 10.6 7.3 8.1 7.3

Width

(A)

3.2 3.2 3.2 3.9 4.0 4.2 4.6 5.5 4.2 6.4 6.7 11.5 8.1

Depth 14.4 12.0 9.7 8.7 7.8 7.0 5 .O 4.0 4.8 3.2 2.4 1.2 1.1

Areddepth’

448 448 448 448 448 448 448,450 450 450 450

448 448

448

-

-

-

+

?

+

?

+ + +

+

+ +

Preferred cytochrome substrate Carcinogenicity

Molecular Dimensions of Substrates, Inhibitors, and Inducers for Cytochromes P-448 (P-450 I) and PB-Cytochromes P-450 (P450 II B)

TABLE 3




18

Dibenz (a. h) rnthracene

Phenobarbital

M a y rapone

Hexobarbital

FIG. 1. Molecular models of substrates, inhibitors, and inducers of the cytochromes P-448 (P450 I) and Pkytochromes P-450 (P450 I1 B). The molecular models of dibenz(a,h)anthracene, 9-hydroxyellipticine, and 7ethoxyresorufin (cytochromes P-448 substrates); and of phenobarbital, metyrapone, and hexobarbital (Phytochromes P-450 substrates) have been plotted by computer graphic determination of the molecular orbitals. Data from Ref, 120.



19

of 85-150 A’ determined for PAH in earlier studies [123]. Determination of the molecular areas of diverse planar chemical structures which are inducers of cytochromes P-448 revealed a similar range of 70-125 A’ [115]. It can be inferred from all these studies that substrates, inducers, and inhibitors of the cytochromes P-448 are all planar molecules, and that any deviations from planarity will diminish selectivity for the cytochromes P-448 and give rise to metabolism by both cytochromes P-448 and PB-cytochromes P-450, and the mixed-type induction. Similarly to the cytochrome P-450 enzyme system, the UDP-glucuronyltransferases also exist as a multigene family; they are also membrane-bound enzymes localized in the endoplasmic reticulum and in the nuclear envelope [ 1241. Again, similar to the substrate preferences seen with cytochromes P-450, the UDP-glucuronyltransferase(s) induced by MC also preferentially metabolize planar phenols such as p-nitrophenol, 4-methylumbelliferone, and 3hydroxybenzo(a)pyrene [125-1281, indicating that MC-inducible proteins, whether hemoprotein or not, interact predominantly with planar molecules. In contrast, the PB-inducible glucuronyltransferases preferentially conjugate bulky, nonplanar molecules such as morphine, chloramphenicol, 4-hydroxybiphenyl, and the hydroxylated metabolite of diphenylhydantoin [ 1291.

G. Substrate Specificities The various cytochrome P-450 proteins not only display different substrate activities, but also display different regio- and stereoselectivities, so that the fate of a chemical in a tissue will be determined not only by the total cytochrome P-450 concentration, but also by the form@) present in that tissue. As the mixed-function oxidase system contributes significantly to the metabolism of almost all xenobiotics, the identification and characterization of the individual forms of cytochrome P-450 involved is of primary importance, since the overall pattern and rates of metabolism will determine the probable toxicity/carcinogenicity of a particular chemical. Although both polyclonal and monoclonal antibodies may be efficient in the identification of individual cytochrome P-450 enzymes in vifrq only chemical substrate probes will allow an assessment of the relative activities of the different cytochromes in viva Initial studies aimed at identifying suitable chemical probes, and using a limited number of “standard” substrates, have largely been unsuccessful because of extensive overlap in substrate specificity among the cytochrome P-450 proteins, and as a result, researchers have become skeptical in using such an approach. However, there is overwhelming and unequivocal evidence testifying to the use of 7-ethoxyresorufin 0-deethylase (EROD) activity to reflect only cytochrome P-448 activity. Extensive studies using purified cytochrome P-450


20

1OA”lDES AND PARKE!

proteins derived from the liver of rats have clearly demonstrated that only cytochrome P-450c, and to a lesser extent cytochrome P-450d, can catalyze this reaction, and no activity has been detected with any other form [130, 131l.Similar observations have been made in rabbit [132]. In the untreated rat, EROD activity appears to be due entirely to P-450d [133]. Good correlation has been obtained between cytochrome P-45Oc determined immunochemically and EROD activity [134]. A major advantage of the EROD assay is that a single product is formed, in contrast to the aryl hydrocarbon (AHH)assay using benzo(a)pyrene as substrate, where a plethora of products are responsible for the increase in fluorescence [ 1351. Furthermore, EROD activity is associated with the murine Ah locus as illustrated in studies conducted in “responsive”and “nonresponsive” strains of mice [136]. A major disadvantage of the EROD assay is that it monitors primarily the P-45Oc isoenzyme [130] and is not sensitive to small changes in P-450d concentration. However, the latter hemoprotein selectively catalyzes the N-hydroxylation of the mutagenic food pyrolysis product Glu-P-1 (1371, and the formation of this product or its resultant mutagenicity in the Ames test may be used to monitor P-450d levels [138]. Another problem associated with such enzymic assays is that the inducing agent, having a long half-life, may be still present in the microsomes tightly bound to the heme and, as a result, the extent of induction may be underestimated. Immunoquantificationovercomes this problem but also suffers from the disadvantage that levels of apoprotein, and not the enzymically functional protein, are determined. A combination of the EROD and Glu-P-1 assays with immunoquantification offers at present the most reliable means for determining cytochrome P-448 activity. To monitor specifically the Phytochromes P-450, another alkoxyresorufin, namely, 7-pentoxyresorufin,is most valuable and appears to be superior to the more frequently used benzphetamine N-demethylase assay [ 137, 1401. The simultaneous determination of different cytochromes in vivo, using a single substrate, such as warfarin, has met with some success as the cytochromes P-450, including P-450c, display regio- and stereoselectivity in hydroxylating this substrate; cytochromes P-448 specifically hydroxylate warfarin at the 6and 8-positions, that is, on the planar moiety of the molecule [134].

H. Metabolic Activation and Detoxication One of the most important differences between the cytochromes P-448 and other families of the hemoproteins, from the point of view of chemical toxicity and disease aetiology, is their contrasting roles in the metabolic activation and detoxication of toxic chemicals and carcinogens. Cytochromes P-446 almost always metabolically activate chemicals to reactive intermediates, that is,

21



electrophiles, which are resistant to subsequent conjugation and interact with intracellular macromolecules and nucleophiles, giving rise to toxicity and carcinogenicity. In contrast, the PB-cytochromes P-450 direct the overall oxidative metabolism of chemicals toward subsequent conjugation and detoxication. The reason for this marked difference is that the cytochromes P-448 accept large planar molecules which they are able to oxygenate in conformationally hindered positions, giving rise to epoxides and other oxygenated metabolites that are poorly acceptable substrates for epoxide hydrolase and other conjugating enzymes, whereas the Pkytochromes P-450 generally oxygenate globular molecules in unhindered positions to form epoxides and other oxygenated products which are readily acceptable substrates for epoxide hydrolase and other conjugases, thereby resulting in detoxication (see Fig. 2). Among the various classes of chemicals known to be selectively activated by the cytochromes P-448, with the formation of reactive intermediates, are the PAH, the aromatic amines and amides, the planar polyhalogenated biphenyls and other halogenated polycyclics, azo compounds, mycotoxins, paracetamol,

PHASE I

PHASE IIC METABOLISM

METABOLISM

-

Excretic

conjuqat ions

Metabolites non-hindered oxygenation

/

I

- - - - - -

Chemical

- - -

Conjugates

-

DC TOXICATION

-

\

- -

-

- - -

ACTIVATION

hindered oxygenation Reactive Intermediates

I

CSH, proteins, thiol enzymes, D N A

Covalent Binding Toxicity and Carcinogenicity

FIG. 2. Xenobiotic metabolism by alternative pathways of detoxication and activation.


22

1OA"IDES AND PARKE

and 4-ipomeanol [15]. However, there are a few known instances where PB induction increases chemical toxicity, or the PB-cytochromes P-450 specifically metabolize chemicals via toxic pathways, and these include the activation of cyclophosphamide, and the toxicity of bromobenzene and carbon tetrachloride. Furthermore, the cytochromes P-448 do not appear to catalyze the metabolic activation of the carcinogenic nitrosamines, which is associated instead with the alcohol-inducible cytochrome P-45Oj (P450 I1 E) and PB-cytochromes P-450 [141-1431.

I. Metabolism of Endogenous Substrates

The cytochromes P-450 hydroxylate steroids regio- and stereoselectively, and individual constitutive cytochrome P-450 proteins that efficiently catalyze specific steroid hydroxylations have been isolated from livers of both rats and rabbits [12, 144,1451. The PB-cytochromes P-450 and cytochromes P-448 can hydroxylate steroids, but generally do so at rates well below those occurring in crude microsomes, indicating that steroids are unlikely to be the primary endogenous substrates of these cytochromes. However, even in the hydroxylation of steroids, PB-cytochromes P-450 and cytochromes P-448 again show distinct differences in regioselectivity with virtually no overlap. For example, cytochromes P-45Oc and P-450d hydroxylate testosterone at the conformationally hindered 6f%position, while cytochromes P-45Ob and P-450e hydroxylate the same substrate in the unhindered 16a-, 168, and 17-positions [ 146, 1471; cytochrome P-450a, enhanced by both the PB and MC groups of inducing agents, selectively catalyzes the 7a-hydroxylation, and to a lesser extent the 6a-hydroxylation, of testosterone [146, 1471. In the 2-hydroxylation of 17gestradio1, cytochrome P-450d is markedly more active than any other cytochrome P-450 protein [148]. The regioselectivity of cytochromes P-448 and PB-cytochromes P-450 is also evident in the metabolism of prostaglandins. The total prostaglandin hydroxylase activity of rabbit liver LM, was greater than that of LM,;LM, hydroxylated prostaglandins primarily at the (o-1)-position and to a lesser extent at the o-position, but these hydroxylations required the presence of cytochrome b, [149]; in contrast, LM,and LM, hydroxylated prostaglandins at the (o-1)-position, LM, showing especially high activity, but did not form any o-hydroxylated metabolites [149]. Subsequent studies established that LM, also readily metabolizes prostaglandins at the (~-2)-position,indicating that this may be one of the major endogenous substratesof this isozyme [150]. Similarly,



23

rat liver microsomes hydroxylated PGE, and PGE, at w- and (w-1)-positions with traces of (~-2)-hydroxylation;pretreatment with MC markedly stimulated (~-2)-hydroxylation,with minimal changes in the other hydroxylations [ 1511. Differences in the sites of oxygenations by the cytochromes P-448 and PB-cytochromes P-450 are also seen with the endogenoussubstrate arachidonic acid. Cytochrome P-450 proteins from livers of rats and rabbits pretreated with p-NF formed primarily w- and (w-1)-hydroxylated products, while, in contrast, preparations from animals pretreated with PB gave primarily four epoxides [1521. A cytochrome P-450 protein catalyzing the epoxidation of arachidonic acid has recently been isolated from human liver [153]. Interestingly, this protein had high catalytic activity toward 7-ethoxyresorufin and the authors suggested that it may be related to cytochromes P-448.

J. Regulatory Control Cytochrome P-45Oc and P-450d appear to be coinduced by a number of xenobiotics including PCB [21], PAH, and methylenedioxybenzenes [32]. However, the levels of P-45Oc and P-450d are increased to different extents by various inducing agents, indicating that they are not under coordinate control; for example, safrole and 3,4,5,3', 4', 5'-hexachlorobiphenyl induce primarily P-450d; whereas MC, other PAH, and p-NF induce mostly P-450c, and Aroclor 1254 induces both forms to the same extent. Furthermore, the mRNAs encoding for P-45Oc and P-450d were found to have different stabilities and to display different induction kinetics, confirming that they are not under coordinate regulatory control [73]. Moreover, when MC-induced hepatocytes were placed in culture, P-45Oc was relatively stable while P-450d was rapidly lost . Cytochrome P-450c, in contrast to cytochrome P-450d, is also expressed extrahepatically [155-1581. Similarly, in mouse the two isozymes of cytochromes P-448 differ in respect of the response to common inducing agents, development,and tissue specificity [155]. In the pregnant rabbit, TCDD induces LM, in the neonate, but in the adult animal, although LM, is induced, the predominant form is LM4[ 1591. In human subjects, immunoblot analysis of 14 liver preparations showed that all samples contained proteins immunologically related to cytochrome P-450d, but only one contained a protein related to cytochrome P-45Oc [160]. The PB-inducible cytochromes P-450b and P-450e are also coinduced but, in contrast to the cytochromes P-448, they are under coordinate regulatory control, the level of P-45Ob being about twice as high as that of P-450e [38,39, 1611. Recent studies, however, revealed that the regulation of these two isoenzymes may be tissue specific [162]; in rat intestinal mucosa, the mRNA


24

IOANNIDES AND PARKE

level of P-450b, but not P-450e, was induced by a number of PB-type inducing agents, while both were increased in the liver.

K. Hemoprotein Turnover The turnover rates of cytochromes P-450b and P-45Oc in normal rat liver, using H'4C0,-labeling to minimize anomalies from extrahepatic amino acid utilization, were found to differ from each other, cytochrome P-45Oc having a shorter half-life. With radiolabeled 5-aminolevulinic acid, the heme turnover was the same for both isozymes, indicating that the turnover of the heme was faster than the apoprotein in the case of P-450b whereas both the heme and apoprotein moieties of cytochrome P-45Oc have the same turnover rates [ 1631. However, in Aroclor-treated animals the turnover rates of the heme and apoprotein moieties of cytochromes P-450b and P-450e were identical to those of cytochrome P-45Oc [164]. In contrast, other workers reported no changes in the rates of degradation of rat cytochromes P-450b and P-450c, or of 5 other cytochrome P-450 proteins, and these were also not affected by pretreatment of the rats with either P-NF or PB [ 1651; they observed a more rapid degradation of the heme moiety than the apoprotein with all proteins, which, in the case of cytochrome P-450b, agrees with the findings of Sadano and Omura [ 1631. The stability of the cytochromes may be altered by the formation of complexes with various ligands. The formation of complexes of cytochromes P-448 with a metabolite of safrole, probably a carbene, following the incubation of liver microsomes from a MC-induced rat with safrole, enhanced the stability of the induced hemoproteins more than it did those induced by PB [166, 1671. Moreover, isosafrole, which forms similar complexes, inhibited the degradation of cytochrome P-450d in addition to enhancing its gene transcription [154,168]; isosafrole also increased the half-life of cytochrome P-450d in isolated hepatocytes [154]. Similarly, 0-NF-induced rat cytochromes P-448, following partial hepatectomy, specifically enhanced the N-demethylation of NN-dimethyl-4aminoazobenzene (DAB),which was not depressed during subsequent liver regeneration; in contrast, PB induced all pathways of DAB metabolism and these were depressed during regeneration, indicating that the p-NF-inducible forms of the cytochrome are more stable [169]. The organotin compound tricyclohexyltin hydroxide prevented the PB-mediated induction of the cytochromes P-450, but had a much less pronounced effect on the induction of the cytochromes P-448 by P-NF or MC [170]; this might be due to the higher stability of cytochrome P-448, although an effect on the induction process cannot be ruled out. The two isoenzymes of the cytochromes P-448 have different stabilities in



25

the presence of certain toxic agents. Treatment of MC-induced rats with CCI, destroyed almost all of the hepatic P-450d but had no effect on hepatic P-45Oc; furthermore,the CCI, treatment did not destroy the induced P-45Oc in rat kidney [171]. Increased cytochrome P-450 stability through ligand formation is not limited to the MC-inducible forms, as the macrolide troleandomycin increases the PCN-inducible form(s) of cytochrome P-450 by inhibiting degradation [ 1721; a metabolite of the antibiotic forms a stabilizing complex with the cytochrome, in a manner similar to that seen with safrole.

L. Hepatocellular and Pulmonary Distribution Hepatocytes are a nonhomogeneous population of cells with respect to mixed-function oxidase activity, reflecting a differential distribution of the various isozymes. Both cytochromes P-448 and PB-cytochromes P-450 are primarily encountered in the centrilobular hepatocytes but their distribution differs in midzonal and periportal hepatocytes, indicating that each form has a unique pattern of distribution. Cytochromes P-448 are uniformally distributed in the hepatic midzonal and periportal regions, but PB-cytochromes P-450 are significantly higher in the midzonal region [ 1731. Isoenzyme distribution may be a major factor in determining the localization of the pathological effectors of hepatotoxins. Large doses of paracetamol, a drug whose activation is mediated by cytochromes P-448 [174, 1751, give rise largely to centrilobular necrosis which may extend to the periportal through midzonal regions. In the lung, both cytochromes P-448 and Pkytochromes P-450 are present in the Clara and type I1 alveolar cells [176], thus explaining why these cells are susceptible to the toxicity of chemicals [177].

V. METABOLIC ACTIVATION OF CHEMICALS Many aspects of chemical toxicity, mutagenicity,and carcinogenicity are due to the oxidative metabolism of chemicals to reactive intermediates which are not readily conjugated and detoxicated and thus react covalently with proteins, enzymes, DNA, and other cellular macromolecules leading to the formation of neoantigens, the inhibition of enzymes, DNA adduct formation, and receptor activation, which may result in immunotoxicity,tissue necrosis, mutations, and malignancy, respectively. This activation of toxic chemicals and chemical mutagens/carcinogens is catalyzed primarily by the cytochromes P-448 (see Table 4), in contrast to the other families of the cytochromes P-450 which mostly catalyze oxygenations that lead to detoxication.

26

IOANNIDES AND PAR=


TABLE 4 Role of Cytochromes P-448 (P450 I) in the Activation of Chemicals Type of reaction involved

Chemical group

Example

Arene oxidation

Benzo(a)pyrene

Arene oxidation?

3,4,3’,4‘-Tetrachlorobiphenyl 4-hinobiphenyl IQ (2-amino-3-methylimidazo[4,5-flquinoline) 2-Acetylaminofluorene N-Methylaminoazobenzene Aflatoxin B, Paracetamol a-Naphthoflavone Ipomeanol

Polycyclic aromatic hydrocarbons Poly halogenated biphenyls Aromatic amines Heterocyclic amines

N-Hydroxylation N-Hydroxylation

Aromatic amides Aminoazobenzenes My cotoxins Drugs Flavonoids Furans

N-Hydroxylation N-Hydroxylation Arene oxidation N-Oxidation Arene oxidation? ?

Various groups of chemical carcinogens may be activated by cytochrome P-448, including those discussed in the following subsections.

A. Polycyclic Aromatic Hydrocarbons Polycyclic aromatic hydrocarbons (PAH) not only induce the cytochromes P-448 but are also preferentially metabolized by these enzymes. Benzo(a)pyrene is perhaps the most extensively studied chemical carcinogen whose activation/deactivation pathways have been largely elucidated. During the 1970s Jerina and his colleagues [1781, using polycyclic aromatic hydrocarbons as models, also addressed the nature of the active site of cytochromes P-450 and advanced the “bay-region” concept, using this to predict the carcinogenic potential of PAH compounds. This hypothesispostulated that epoxidation in the bay region of a PAH compound would result in chemical reactivity, and hence in carcinogenicity/mutagenicity, as similarly predicted by molecular orbital calculations for the ease of carbonium ion formation. Solubilized, purified cytochrome P-448 preparations (P-45Oc) were more efficient than the Pkytochromes P-450 in converting benzo(a)pyrene and (~)-truns-benzo(a)pyrene-7,8-dihydrodiol,the precursor of the ultimate carcinogen, into mutagens [179] (see Table 5); similarly, hepatic microsomal S9 preparations from MC-treated animals are markedly more efficient than similar preparations from PB-induced animals in activating benzo(a)pyrene to muta-


27


TABLE 5 Metabolism of (i)-trans-Benzo(a)pyrene 7,8-Dihydrodiol by Reconstituted Cytochrome P-450 Systems Oxygenation products

Cytochrome P-448 P-448 P-450 P-450

Epoxide hydrolase None

+

None t

-

trans 1 cis 1 trans 2 cis 2 Total (nmol formedhmol cytochrome per min) 0.14 0.17 0.005 0.004

0.61 0.60

0.02 0.03

1.03 1.00 0.06 0.06

0.08 0.08 0.004 0.06

2.69 2.77 0.10 0.10

Note. Incubation mixtures contained 0.2 nmol cytochrome P-448 or 0.4 nmol cytochrome P-450,225-450 units NADPH-cytochrome c reductase, 30-50 pg phosphatidylcholine, 34 units epoxide hydrolase, 0.5 pmol NADPH, 3 pmol MgCI,, 100 pmol potassium phosphate buffer pH 6.8, and 40 nmol 'H-(?)trans-benzo(a)pyrene 7,&dihydrodiol in a final volume of 1.O mL. Mixtures were incubated for 10 min at 37°C. Data from Ref. 179.

gens and in converting it to the 7,8-diol-9,10-epoxide [180, 1811. Pretreatment of rats with PB increased the formation of the 4,5-diol of benzo(a)pyrene but had little or no effect on the 7,8- and 9,lO-diol formation; the formation of all three diols was, however, induced by pretreatment with P-NF and MC, the least effect being seen with the 4,5-diol [182]. The MC-induced increase in benzo(a)pyrene mutagenesis was inhibited by 9-hydroxyellipticine, a specific inhibitor of cytochrome P-448 activity [ 1831. Intraperitoneal pretreatment of mice with MC increased the formation of benzo(a)pyrene 7,8-diol in both lung and liver, while no such effect was seen following treatment with PB [184]. Furthermore, pretreatment of mice by intratracheal instillation of benzo(a)pyrene increased markedly the covalent binding of the carcinogen to lung DNA [1851. Supporting these findings are immunological studies which showed that the metabolic activation of benzo(a)pyrene to mutagens was markedly inhibited by antibodies to cytochrome P-45Oc but was unaffected by antibodies against cytochrome P-450b [1861. Similarly, a monoclonal antibody against MCinduced cytochromes P-448 inhibited the conversion of benzo(a)pyrene 7,8-diol to mutagens by liver S9 preparations from MC-induced responsive mice, while no effect was seen with an antibody to PB-induced microsomes [ 1871. Finally, good correlation has been obtained between rat liver cytochrome P-448 content, determined by the EROD assay, and the activation by rat liver S9 preparations of benzo(a)pyrene to mutagens in the Ames test [ 1871. However, in contrast to these findings, Kawano et al. [ 1881, using purified rat liver preparations, found that the PB-inducible form, P-450e, was the most effective in activating


IOANNIDES AND PARKE benzo(a)pyrene to mutagens. Finally the alcohol-inducible cytochrome P-450 isoenzymes (P450 I1 E) appear not to catalyze the activation of PAH [189,190]. The preferential activation of PAH by cytochromes P-448 is not confined to benzo(a)pyrene. Cytochromes P-448 also convert MC, 6-aminochrysene, benz(a)anthracene, dibenz(a,h)anthracene, dibenz(a,c)anthracene, and many other bay-region-containing PAH to highly mutagenic species [ 186, 188, 191-1951. Similarly, their nitrogen-heterocycle analogues induce cytochrome P-448 activity [85] and appear to be activated by the same enzyme [196]. It appears that the cytochromes P-448 are unique in their capacity to oxygenate PAH at the conformationally hindered or bay-region positions, forming epoxides that are not readily further metabolized and deactivated by phase I1 mechanisms [15,197], and as a result interact with cellular nucleophiles such as DNA to initiate the processes of mutagenesis and carcinogenesis. Microsomes from rats treated with PB or nonplanar PCB showed a 4-fold increase in benzo(a)pyrene metabolism, the major increase being in the formation of the 4,5-diol; whereas microsomes from MC- and planar PCB-treated animals showed a 10-fold increase in metabolism, the major increases being in the 7,8- and the 9,lO-diols (i.e., bay region), the precursors of the 7,8-diol-9,10-epoxide, the ultimate carcinogen [1981. Similarly, liver microsomes from MC-treated rats metabolized 6-methylbenzo(a)pyrene at the 3,4-position to an extent of 60% of the total metabolism, while PB-induced microsomes metabolized only 10% at this position, which is associated with the formation of the ultimate carcinogen, 6-methylbenz(a)anthracene-3,4-diol-1,2epoxide [ 1991. Even in non-bay regions, cytochromes P-448 form epoxides more readily than do the Pkytochrornes P-450; cytochromes P-450b and P-45Oc both form 1,Zarene oxides of naphthalene and anthracene, the overall activity being markedly higher with cytochrome P-450c, especially in the case of anthracene [200].

B. Polyhalogenated Biphenyls Cytochromes P-448 selectively catalyze the metabolic activation and binding of the planar PCB isomer 3,4,3',4'-tetrachlorobiphenyl, while the P E cytochromes P-450 were more effective in activating two nonplanar isomers having a chlorine at an ortho position-that is, 2,4,2',4'- and 2,4,2',5'tetrachlorobiphenyls [201]; the planar isomers exhibit much higher toxicity than the nonplanar analogues [202, 2031. Similarly the PB-inducible cytochrome P-450b readily metabolized the nonplanar dichlorobiphenyls where both chlorine atoms were located at ortho positions. In contrast, cytochrome P-45Oc metabolized the planar isomers-that is, where ortho positions were unsubstituted. Finally, both isozymes metabolized the dichlorobiphenyl isomers


29


TABLE 6 Metabolism of Polybrominated Biphenyls with Liver Microsomes from 3-Methylcholanthrene- and Phenobarbital-Induced and Control Rats Rate of metabolism (pmol/mg protein per min) Congener ~

MC-induced

PB-induced

Control

0.33 109.6 100.7 57.6 43.5 0.0 58.8 0.06 0.02 0.05 0.16 0.0

201.7 0.0 0.02 52.8 184.6 66.2 0.01 0.0 23.7 0.03 0.0

0.59 0.13 0.0

~

2,2’-DBB 4,4‘-DBB 3,4,4’-Tri-BB 2,3,3’,4’-Tetra-BB 2,4,2’,5’-Tetra-BB 2,5,2’,5’-Tetra-BB 3,4,3’,4’-Tetra-BB 3,5,3‘,5’-Tetra-BB 2,4,5,2‘,5‘-PBB 2,4,5,3’,4’-PBB 2,3,4,2’,4’,5’-HBB 3,4,5,3‘,4’,5‘-HBB

0.01

-

0.0 0.0

-

Note. microsomal incubations were carried out aerobically at 37°C in 50 mM Tris-HC1 buffer pH 7.5 in the presence of an NADPH-generating system and 1mg liver microsomal protein/mL from MC- and PB-induced, and control rats, to which the PBB congeners were added. Metabolism was determined from the rate of disappearance of substrate. - means not determined. Data from Ref. 205.

with only one chlorine atom in the ortho position [204]. Similarly, in an in v i m study of the metabolism of polybrominated biphenyls, microsomes from MC-treated rats were markedly more effective than the PB-induced in metabolizingthe planar isomers having no ortho substitution,while PB-induced microsomes were more effective in the metabolism of the nonplanar isomers with ortho substitutions on both rings [205] (see Table 6).

C. Aromatic Amines N-Hydroxylation is the first step in the activation of aromatic amines while ring hydroxylation is considered to be a deactivation pathway. The Nhydroxylation is catalyzed by two liver enzyme systems, namely, the FAD-monooxygenase system and the cytochromes P-448. 4-Aminobiphenyl


30

IOANNIDES AND PAR=

[206,207] and 2-aminofluorene are readily N-Hydroxylated and converted into mutagens by cytochromes P-45Oc and P-450d, but much less readily by cytochromes P-450b and P-450e [ 188, 207, 2081. Liver microsomal preparations from Aroclor 1254- and MC-induced rats activated 2-naphythylamine to mutagens more readily than did PB-induced microsomes [209], and the isoenzyme responsible appears to be cytochrome P-450d [206]; MC-induced enzyme preparations from “responsive” B, mice were more efficient than similar preparations from “nonresponsive” D, mice [210]. Similarly the planar 3, 3’-dichlorobenzidine is selectively activated to mutagens by cytochrome P-450d [94] despite the fact that both cytochrome P-448 isoenzymes were induced by the amine. The activation of 2-aminoanthracene to mutagenic intermediates is catalyzed by rabbit LM, but not LM, [211], and the binding of 2-aminoanthracene to murine lung DNA is increased by pretreatment of the animals with benzo(a)pyrene [1851. Interestingly, the mutagenicity of some aromatic amines such as 2-aminofluorene and 4-aminobiphenyl was enhanced also by dietary exposure of animals to high levels of alcohol [189, 1901. The heterocyclic aromatic amine premutagens, formed during the pyrrolysis of proteins and amino acids, are also activated to mutagens by N-hydroxylation catalyzed by the cytochromes P-448 but not by PB-cytochromes P-450 [141, 212,2131. The mutagens Trp-P-1, Glu-P-1, Trp-P-2, and IQ were found to be activated by cytochromes P-448, particularly the high-spin form cytochrome P-450d [137, 138, 214-2161. Furthermore, the mutagenicity of Trp-P-2 was inhibited 85% by antibodies to cytochrome P-448, but only 8%by antibodies to PB-cytochromes P-450 [217].

D. Aromatic Amides The metabolic activation of the aromatic amide 2-acetylaminofluorene (2-AAF) has been studied extensively. As with the corresponding amine, its activation proceeds through N-hydroxylation which is catalyzed exclusively by cytochromes P-448 [131, 2181, confirming previous observations where antibodies to cytochromes P-448, but not PB-cytochromes P-450, decreased the N-hydroxylation and covalent binding of the carcinogen [219]. Pretreatment of animals with 2-AAF or MC increases the N-hydroxylation of the amide, and increases the cytochromes P-448 content as determined by EROD activity [181, 2201. Similarly, rabbit liver LM4 catalyzes the N-hydroxylation of 2 - M , whereas LM, and the PB-inducible LM2 did not, although the former could hydroxylate the amide at the 7-position [221]. Furthermore, monoclonal antibodies to MC-induced cytochromes P-448 inhibited the N-hydroxylationof 2-AAF [222], and the mutagenicity of 2-AAF was inhibited 60% by antibodies to cytochrorne P-448 but only 35% by antibodies to cytochrome P-450 [186,



31

2191. The mutagenicity of 2-AAF catalyzed by control and MC-induced liver microsomal preparations was similarly inhibited by a monoclonal antibody to cytochromes P-448 but not by a monoclonal antibody to Phytochromes P-450 [187]. Furthermore, the mutagenicity of 2-AAF was enhanced in “responsive” mice by pretreatment with MC, whereas the same treatment had no effect in “nonresponsive” mice [223]. Cytochromes P-45Oc and P-450d have somewhat different substrate specificities, P-45Oc being efficient in forming arene oxides of PAH, while P-450d is especially active in the N-hydroxylation of heterocyclic and aromatic amines. Cytochrome P-45Oc readily catalyzes the metabolism of benzo(a)pyrene, 7-ethoxyresorufin, and 7-ethoxycoumarin while cytochrome P-450d preferentially metabolizes acetanilide [109, 2241, although other studies have indicated that P-45Oc is more efficient that P-450d in acetanilide hydroxylation in reconstituted systems [225]. Cytochrome P-450d is considerably more active in the hydroxylation of aromatic amines.

E. Azo Compounds The N-hydroxylation of N-methylaminoazobenzene and the metabolic activation of N-dimethylaminoazobenzeneare catalyzed by cytochromes P-448 [226, 2271; cytochrome P-450d catalyzes the N-hydroxylation of N-methylaminoazobenzene at twice the rate of cytochrome P-450c, although the latter is more active in ring hydroxylation [227]. The activation of o-aminoazotoluene to mutagens was inhibited more by antibodies to cytochrome P-448 than by antibodies to Phytochrome P-450 [1861; when purified cytochromes were used as activation systems, only cytochrome P-45Oc was active in metabolizing the aminoazotoluene to mutagens [ 1881. In contrast, the mutagenicity of 3-methoxy-4-aminoazobenzenewas increased to a larger extent by pretreatment with PB than MC [89].

F. Aflatoxin B, The activation of this mycotoxin to mutagenic and carcinogenic products, involving epoxidation of the 2,3-double bond and possibly other oxygenations, appears to be catalyzed by the cytochromes P-448 and possibly other forms of the cytochromes. A comparison of 5 purified rat cytochrome P-450 proteins in the activation of aflatoxin B, into mutagens showed that cytochrome P-450d and a sex-related male form were the most efficient, although significant activity was also seen with cytochromes P-45Oc and P-450e [228]. The high efficiency of cytochrome P-450d has been documented by other workers, who further showed that although PB-inducible forms also activated this mycotoxin,


32

IOANNIDES AND PARKE

they did so to a much less extent [216]. In other studies using reconstituted systems, both cytochromes P-448 and PB-cytochromes P-450 activated aflatoxin B, to a DNA binding species [216, 2291. In studies on the activation of aflatoxin by liver preparations from responsive and nonresponsive mice, mutagenicity was increased by PB treatment in both mouse strains and by MC only in the responsive mice [187,230]. However, the mutagenicity of aflatoxin by MC-treated responsive mice could not be inhibited by a monoclonal antibody to the PAH-inducible forms of P-450. It is possible that another MC-inducible form of P-450, not sharing the epitopes recognized by this monoclonal antibody, may be responsible for the mutagenicity of aflatoxin B, in MC-induced responsive mice. Such a protein, induced by MC, with an absorption maximum at 448.5 nm and distinct from rat cytochrumes P-45Oc and P-450d, has been isolated and purified from hamster liver, and may possibly represent a new isozyme within the P450 I family [231]. Surprisingly, this protein was specific to hamster and could not be induced in other animal species [232]. Interestingly, it has recently been demonstrated that treatment of male rats with inducers of the P450 111 family, such as PCN and dexamethasone, increased the 9-hydroxylation of aflatoxin B, and enhanced its mutagenicity in the Ames test [233]. G. Drugs and Other Chemicals The role of cytochromes P-448 in the generation of reactive intermediates is not confined to chemical carcinogens, for drugs such as the analgesic paracetamol may be converted to covalently bound products by cytochromes P-448, but not PB-cytochromes P-450, in both rat and rabbit [174, 1751. The alcohol-inducible rabbit LM3, is also effective in activating this drug [ 1751. Similarly, cytochromes P-45Oc and P-450d catalyze the 6-hydroxylation of the toxic muscle relaxant drug zoxazolamine, while cytochromes P-450b and P-450e are poor hydroxylators of this drug [147]. Furthermore, a good correlation exists between the murine cytochrome P,-450 metabolism of this drug and shorter paralysis time [234]. Similarly, the furan 4-ipomeanol is activated by MC-inducible cytochromes P-448 [235], and although both PB and MC stimulate 4-ipomeanol metabolism, the former enhances mostly detoxication pathways, while the latter enhances the formation of intermediates covalently binding to liver [235, 2361. In contrast, the anesthetic fluroxene is more toxic when rats are pretreated with PB than when pretreated with MC, benzo(a)pyrene, or P-naphthoflavone [237, 2381. Although these studies indicate that PB-cytochromes P-450 may selectively metabolize fluroxene to a reactive intermediate,cytochromes P-448 are very susceptible to destruction by fluroxene metabolites, and it is possible



33

that the cytochromes P-448 catalyze the formation of an intermediate which leads to their destruction [238]. The cytotoxic drug cyclophosphamide is also preferentially metabolized and activated by Pkytochromes P-450 since its activation to mutagens was markedly increased by induction with PB but not MC [239], and metabolism to embryotoxic compounds in vitro was more readily catalyzed by S9 activation systems derived from PB-treated animals [240, 2411.

H. Flavonoids PB pretreatment of rats decreased the metabolism of the flavonoids a-naphthoflavone (a-NF) and p-naphthoflavone (f3-NF) by liver microsomal preparations, while pretreatment with MC or p-NF enhanced the metabolism of f3-NFbut decreased that of a-NF [242]. Neither substrate was metabolized by purified cytochrome P-450b, but cytochrome P-45Oc metabolized both substrates. The pattern of metabolites was unaffected by PB pretreatment, but MC or p-NF caused considerable changes stimulating the formation of the 5,6 and 7,8-diols of p-NF, and the 73-diol of a-NF [242, 2431. Finally, the clastogenicity of a-NF in Chinese hamster ovary cells was induced by TCDD, but not PB; TCDD also stimulated the metabolism of a-NF and especially the formation of the 7,8-diol [244].

I. Nitrosamines Cytochromes P-448 appear not to play a major role in the bioactivation of nitrosamines to mutagens. The mutagenicity of dimethylnitrosamine (DMN) was not increased when the activation systems were derived from rats pretreated with the cytochrome P-448 inducer Aroclor 1254 [245], and purified Phytochromes P-450 and cytochromes P-448 did not activate DMN to mutagens, despite their being able to catalyze its demethylation, which is generally accepted as the rate-limiting step in the activation of DMN [246]. The mutagenicity of N-nitrosomorpholine was similarly not enhanced by PB or MC pretreatment of mice [230], and monoclonal antibodies to PB- or MC-induced cytochrome P-450 proteins did not inhibit the mutagenicity [187]. More recent studies have shown that of 11 purified rat cytochrome P-450 proteins, only the ethanol-inducible cytochrome P-45Oj (P450 I1 E) could demethylate DMN at concentrationsof 0.5 and 5.0mM [247]. It has been suggested that cytochrome P-45Oj may be responsible for all the DMN-demethylase activity at substrate concentrations which are relevant to the hepatocarcinogenesis of the nitrosamine [35]. The activation of other nitrosamines also appears to be catalyzed by the isoenzyme of cytochrome P-450 which is inducible by alcohol, and

IOANNIDES AND PAR=


34

activation systems derived from the livers of alcohol-treated animals were more efficient than controls in converting DMN and nitrosopyrrolidine to mutagens [142, 1431. In recent studies employing reconstituted systems with purified cytochrome P-450 proteins, the a-hydroxylation of N-nitrosopyrrolidine, considered to be the rate-limiting step in its activation, was catalyzed preferentially by the alcohol-inducibleLM,, at low carcinogen concentrations [248]. The metabolic activation of another cyclic nitrosamine, namely nitrosopiperidine, was stimulated by pretreatment of rats with PB but not MC [ 1411.

J. TCDD This toxin was resistant to metabolism by microsomal preparations [249], but freshly isolated hepatocytes from hamster and rats were able to convert TCDD to polar metabolites. Metabolism was markedly enhanced in both species by pretreatment with MC or TCDD itself, and to a lesser extent by isosafrole, but PB had no effect, indicating that cytochromes P-448 selectively metabolize this toxin [250].

VI. MECHANISM OF ENZYME INDUCTION: THE Ah LOCUS Inducers of the cytochromes P-450 stimulate the synthesis of new proteins primarily by enhancing their rates of transcription. This mechanism appears to be common to all cytochrome P-450 proteins but does not exclude the presence of additional alternative mechanisms. Posttranscriptional regulation has been suggested in the case of the alcohol-inducible P-45Oj protein [60, 2511.

A. The Ah Receptor The elegant work of Nebert and his colleagues with inbred strains of mice has contributed enormously to the elucidation of the complex process which results in the induction of the cytochromes P-448 [81,252]. A receptor, the Ah receptor, has been demonstrated in the hepatic cytosol of mice which binds avidly, but noncovalently, with the inducing agent and which is associated with the induction of cytochromes P-448 [253]; this hydrophobic receptor appears to be composed of two subunits but contains only one binding site [254] and the binding appears to involve one or more sulfhydryl groups whose integrity is essential [255]. It appears that it is the parent chemical rather than a reactive intermediate or metabolite that interacts with the receptor [256].In addition to the ligand binding, a DNA-binding domain exists on the receptor [257, 2581. The inducer-receptor complex translocates into the nucleus where it activates



CONJUGATES

/

-

35

EXCRETION

conlugasas

I N ACTlV E METABOLITES

cytochrome P-450

0 ETOX IC AT10 N

cocarcinogenesis DNA- t ranscription

FIG.3. The roles of cytochromes P-448 (P450 I) and the Ah receptor in the formation of reactive intermediates and the mechanisms of chemical carcinogenesis.


36

1OA”IDES AND PARKE

structural genes leading to increased transcription and synthesis of the Ah receptor protein, menadione reductase, protien kinase c, other enzymes, and the cytochrome P-448 apoproteins, which in the cytosol interact with heme to form the P-448 holoenzymes (see Fig. 3). A good correlation exists between the amount of complex translocated into the nucleus and the amount of P450 I mRNA induced [259]. Interaction of the inducing agent with the receptor increases the affinity of the receptor for DNA [257, 2601. Many endogenous compounds, including steroid hormones, failed to interact with the receptor, and there is considerable evidence indicating that none of the characterized steroid receptors is identical to the Ah receptor [261-2631. It is probably that, as with the hormone receptors, the Ah receptor is of nuclear origin, and that the presence of the Ah receptor in the cytosol is due to nuclear leakage during subcellular fractionation [260, 2641, hence what is measured is not translocation of the inducer-receptor complex into the nucleus but binding to chromatin [265]. In strains of mice refractive to cytochrome P-448 induction, only a defective, poor-affinity receptor is detectable, whereas a functioning receptor is always present in responsive mice [266, 2671. TCDD, the most potent inducer of cytochrome P-448 activity, binds avidly to the Ah receptor and its displacement has been used to assess the affinity of other chemicals for the receptor; being poorly metabolized, TCDD displays a long half-life and is thus capable of producing sustained effects in the cell [268, 2691. In accordance with the role of a receptor protein, cytochromes P-448 are induced following administration of very small doses of the inducing agent; a single dose of 50 pgkg of benzo(a)pyrene [ 1321,or a single intraperitoneal dose of 500 pgkg of 2-AAF or safrole [lo41 substantially increased the hepatic EROD activity in rats. The Ah receptor in rodents is not confined to the liver but has been found in other tissues, including the lung, kidney, intestine, brain, spleen, testes, skin, and thymic tissues. In man, the receptor has been detected in lymphocytes[270], placenta [271], and lung [272].

B. The Ah Receptor and Immunotoxicity The immunotoxicity associated with some polycyclic aromatic hydrocarbons is also believed to be mediated through the Ah receptor. PAH which are carcinogenic also possess immunosuppressive activity, whereas noncarcinogens such as benzo(e)pyrene and anthracene have no immunotoxic effect [273]. “Responsive” strains of mice are more susceptible than “nonresponsive” mice to the immunotoxic and carcinogenic effects of the PAH [252]. Coplanar polybrominated biphenyls (PBB), which induce the cytochromes P-448, elicit immunotoxic effects in responsive but not in nonresponsive mice [274], whereas the nonplanar diorthosubstituted PCB, which induce the PB-



37

cytochromes P-450, have no immunosuppressant effect in either strain of mice [275,276]. Other cytochrome P-448-inducing agents, such as TCDD, exert their respective inmunotoxic and teratogenic effects through the Ah receptor [277-2791, and the induction potentials of individual chlorinated dibenzo-pdioxins correlate with their toxicity [280, 2811. a-Naphthoflavone antagonizes both the cytochrome P-448 inductive and immunosuppressive effects of TCDD in cultured murine splenic lymphocytes by competing with TCDD for the Ah receptor [282]. Recent studies have suggested that interaction with the Ah receptor may not be sufficient for the immunosuppressive activity of PAH, as 1,2,3,4-dibenzanthracene induced cytochromes P-448 but did not manifest immunotoxicity [283]. Furthermore, 7,12-dimethylbenz(a)anthracene induces its immunosuppressive effect in both responsive and nonresponsive strains of mice [284]. Two rat strains with markedly different susceptibility to the lethal effects of TCDD were equally responsive to cytochrome P-448 induction and had a similar number of Ah receptors which bound TCDD to a similar degree [285]. Similarly in the guinea pig, no induction by TCDD occurs, although this species is very sensitive to the toxicity of the chemical [286].

C. Ah Receptor Binding and Cytochrome P-448 Induction The relationship between the Ah receptor and cytochrome P-448 induction is affirmed by the good correlation that exists between the induction of AHH or EROD activities by halogenated aromatic hydrocarbons, such as biphenyls, dibenzofurans, dioxins, azoxybenzenes, azobenzenes, PAH, and ellipticines, and the avidity with which they bind to the Ah receptor [234, 235, 280, 287-2891. Furthermore, tar particulates from cigarette smoke contain compounds with affinity for the Ah receptor [290], and administration of cigarette smoke tar to animals enhances EROD activity (unpublished observations). Of the PCB, the coplanar isomers display the highest affinity for the receptor and are also the most potent cytochrome P-448 inducers [86]; loss of planarity resulting from the introduction of a chlorine atom in the ortho position not only decreased the extent of cytochrome P-448 induction but also lowered its affinity toward the receptor. Excellent correlations have been demonstrated between the molecular dimensions of PCB and their binding to rat liver Ah receptor and the induction of cytochromes P-448 [1201 (see Fig. 4.). Similarly, polychlorinated terphenyls which induced EROD activity in the liver of rats also displaced TCDD from the cytosolic receptor [291]. a-Naphthoflavone, like TCDD, binds to rat and murine hepatic Ah receptor, but unlike TCDD, it does not induce cytochrome P-448 activity. fl-Naphthoflavone also binds to the receptor but, in contrast to a-naphthoflavone, it does induce cytochrome P-448 activity and is more planar than the a-isomer [292].

IOANNIDES AND PARKE


38

0 Y,

V w

I

d 9

3

2

3

I

07 4

5

6

7

8

Arca/depth

Area/depth

FIG. 4. Correlation plots of the molecular dimensions of a series of polychlorinated biphenyls with (a) rat TCDD cytosolic receptor binding, and (b) cytochrome P-448 induction in rat hepatoma cells. (a) -log EC,, is for receptor binding; (b) -log ECs0is EROD activity. The PCB are identified as follows: (1) 3,3',4,4',5-penta; (2) 3,3',4,4'-tetra; (3) 2,3,4,4',5-penta; (4) 2,3,3',4,4'-penta; ( 5 ) 2,3,3'4,4',5'-hexa; (6) 2,3,3',4,4',5-hexa; (7) 2,3',4,4',5-penta; (8) 2',3,4,4', 5-penta; (9) 2,3',4,4',5,5'-hexa; (10) 2,3,4,4'-tetra; (11) 2,2',4,4',5,5'-hexa; (12) 2,3',4,4',5',6-hexa; (13) 2,2',4,4'-tetra; and (14) 2,3,4,5-tetra. Data from Refs. 120 and 513. D. Dimensions of the Ah Receptor The exact nature of the cytosolichuclear protein that acts as the Ah receptor remains elusive, but it appears that it shares many structural and physicochemical properties with the steroid receptors [293,294]. It has even been suggested that the glucocorticoid and dioxin receptors are the products of two members of the same gene family [295]. Indirect evidence has provided some information regarding the size of its binding site; good correlationsexist between the spatial dimensions of the isomeric PCB and their affinity for the Ah receptor, and also their ability to induce the cytochromes P-448 [120]. As similar good correlationswere also obtained between the molecular dimensions of substrates and their interaction with the binding site of cytochromeP-448and its induction [ 115, 1161, it may be inferred that the substrate binding sites of the cytochromes P-448 exhibit structural similarities to the Ah receptor binding site. The

9



39

observation that inducers of the cytochromes P-448 possess the same dimensions as substrates lends support to this hypothesis. Planar molecules which act as potent cytochrome P-448 inducers exhibit a length and width of about 14 8, and 8 A, respectively [116], similar to the optimum dimension of 13.7 X 6.8 8, found for indoles that are potent ligands of the Ah receptor [296]. However, there was no correlation between binding avidities to rat liver Ah receptor and cytochrome P-448 induction potency (increased AHH and EROD activities) for a series of PAH [297], or for a series of 26 polychlorinated dibenzofurans [91].

E. Regulation of Cytochrome P-450d Although induction of cytochrome P-45Oc appears to be regulated through the Ah receptor, cytochrome P-450d is also formed, and is induced to the same extent in both responsive and nonresponsive mice [298], indicating that factors other than the Ah receptor are important in the regulation of cytochrome P-450d induction.

F. Species Differences Although studies of the Ah locus have mostly been conducted in inbred strains of mice, the presence of the receptor protein has also been demonstrated in other laboratory animals and man [280, 299-3011; rat and mouse Ah receptors are similar but not identical [302], and these receptors may have different affinities for certain ligands [272]. Using a QSAR approach employing 7-substituted 2,3-dibenzo-p-dioxins,marked differences in their binding to the receptors of various mammals were observed, indicating the heterologous nature of the receptor [303]. However, the concentration and avidity of binding to the Ah receptor cannot explain the species differences in cytochrome P-448 induction. Furthermore, PB treatment of “responsive” mice and rats, which increases the hepatic concentration of the Ah receptor, did not result in enhanced induction of cytochrome P-448 activity when the animals were induced with appropriate agents, indicating that Ah receptor levels are not rate limiting in the induction process [304]. The CH3/HeJ strain of mice are more susceptible to benzo(a)pyrene-induced tumorigenesisthan the C57BW6J strain, although the latter possessed 4 to 6 times more cytosolic receptor [305]. Human breast carcinoma and rat hepatoma cell lines show high inducibilities despite having small amounts of Ah receptor [306, 3071. Accidental exposure of humans in Taiwan to rice contaminated with PCB led to increases in cytochrome P-448 activity; placental AHH activity was markedly stimulated but the concentrations of Ah receptor remained low, indicating that low concentrations of receptor are capable of mediating marked increases in cyto-


40

IOANNIDES AND PARKE

chrome P-448 activity [308]. 2,2-Dimethyl-5-tert-butyl-l,3-benzodioxole (DBBD)antagonizes MC but not PB induction in responsive mice [309], probably because it decreases the levels of receptor available for binding [310].

G. Further Aspects of the Ah Locus Taken as a whole, the foregoing studies indicate the importance of the Ah locus in mediating the carcinogenicity/toxicity of exogenous chemicals. However, recent studies have shown the Ah locus may be much more complex than initially envisaged. Hepatic cytosolic Ah receptor concentrations, and several enzymes associated with the Ah locus, have been correlated with induction by P A H in responsive and nonresponsive mice, and in recombinant inbred lines; induction of AHH by M C could be achieved, however, in mice phenotyped as nonresponsive [311]. If formation of the inducer-receptor complex is inadequate for full induction of cytochromes P-448,it is possible that the gene transcription is modulated by a further mechanism [312]. Additional genes may also determine whether an inbred strain of animal will be susceptible or resistant to certain carcinogens; for although the C3H and C57BW6 strains of mice are both “responsive,” they exhibit a 5-fold difference in benzo(a) pyrene carcinogenicity [313].TCDD induces the synthesis of the low-spin cytochrome P-448 isoenzyme in a human cell tumor line which appears devoid of the receptor [304].Finally, the role of the actual levels of the cytosolic receptor in the induction process remains unclear; cytochrome P-448 induction by PCB was not dependent on the Ah receptor levels [314],although in other studies in responsive mice, a decrease in receptor binding was accompanied by similar decreases in cytochrome P-448induction [3lo]. In the fetus a different situation exists in that the levels of Ah receptor appear to be rate limiting in the induction of hepatic P450 I proteins by MC [315]. Another cytosolic protein (4s)isolated from rat liver-which binds PAH,but not TCDD, is distinct from the Ah receptor, and does not interact with steroids or retinoids-was believed to have a role in the expression of cytochrome P-45Oc [316-3181,but recent studies indicate that it has no major role in P-448 induction [289,3191. It is present in many tissues [320]and in contrast to the Ah receptor, it binds benzo(e)pyrene [321,3221,which is not associated with cytochrome P-448 induction [ 141, providing additional evidence that this protein plays no major part in the induction process. It has been suggested that its function may be as a carrier protein for polycyclic aromatic hydrocarbons [323,3241. However, more recent work has established that the 4s protein interacts with regions of the rat cytochrome P-45Ocgene and may have arisen in the expression of this cytochrome [321,325,3261. Compounds which interact avidly with the Ah receptor but fail to induce cytochrome P-448activity may act as antagonists decreasing the induction



41

potency of inducing agents; for example, 2,4,6,8- and 1,3,6,8-tetrachlorodibenzofurans bind to the rat Ah receptor but are poor inducers of cytochrome P-448 activity in rat hepatoma cells in culture, but when coadministered with TCDD, significantly decrease the inductive potency of the latter [327]. The methylated analogue 6-methyl-1,3,8-trichlorodibenzofuranis an effective antagonist of TCDD; it does not induce cytochromes P-448 when administered to rats but decreases the inductive potency of TCDD, both as measured by the EROD assay and by immunoquantification, indicating that it acts as a competitive inhibitor, preventing the binding of TCDD to the Ah receptor [328]. As induction of cytochrome P-448 activity is closely associated with carcinogenicity,Ah receptor antagonists may have potential use as anticarcinogens. Glucorticoids appear to play a role in the regulation of cytochrome P-448 induction, for although dexamethasone itself does not induce AHH activity in isolated hepatoma cells, it did potentiate the effect of 1,Zbenzanthracene [329]; similar effects were seen in human fetal liver hepatocyte cultures [330] and in hepatocyte cultures from adult and fetal rat livers [331,332]. Potentiation of the cytochrome-P-448-inductive effect of PAH might be mediated by the glucocorticoid receptor [330] but the mechanism remains unclear. Glucocorticosteroids added to rat liver microsomal preparations enhanced the 2-hydroxylation of biphenyl, a cytochrome-P-448-mediated reaction [333]; but no such increase was seen in EROD activity (unpublished observations). A labile protein, responsible for repressing the transcription of the cytochrome P450 I A1 gene, has been proposed [334] and was recently confirmed [335] from observations of “superinduction” of gene expression in vivo, by RNA hybridization following partial inhibition of protein synthesis by cycloheximide, after administration of the inducing agent, MC. No receptor has been identified for the other type of inducers, and the mechanism through which these agents trigger an accelerated transcription of the Pkytochrome P-450 genes remains unknown.

VII. CYTOCHROME P-448 AND CHEMICAL TOXICITY

As cytochromes P-448 primarily activate chemicals, it might be expected that animal species, or particular tissues, exhibiting high cytochrome P-448 activity would be more susceptible to the toxicity of chemicals, as increased amounts of reactive intermediates would be formed, which consequently would overwhelm the detoxication pathways, resulting in covalent binding to cellular nucleophiles and ultimate tissue damage. If the chemical is also an inducing agent of the cytochromes P-448, such as the PAH, then the toxicitykarcinogenicity following repeated administration will depend, not only on the basal


42

IOANNIDES AND PARKE

constitutive cytochrome P-448 activity but, more importantly, on the extent of the induction of this hemoprotein. A number of studies carried out primarily in inbred strains of Ah “responsive” and “nonresponsive” mice are in agreement with this [336]. A. Paracetamol The metabolic activation of this drug to its hepatotoxicreactive intermediate, believed to be a benzoquinoneimine, is catalyzed by the MC-inducible cytochrome P-448 but not by PB-cytochromes P-450 [174,175]. Pretreatment of mice with MC increased the paracetamol-induced hepatotoxicity in responsive B6 mice but had little effect in nonresponsive D2 mice [222]. Similarly, pretreatment of mice with MC exacerbated the paracetamol-induced ophthalmotoxicity in responsive mice only, while pretreatment with PB offered protection [337, 3381. Similar observations have been made in other animal species. Pretreatment of hamsters with MC potentiated paracetamol hepatotoxicity and increased the mortality associated with large doses while, in contrast, pretreatment with PB had a protective role [339]. The hamster, in contrast to the rat, shows marked sensitivity to paracetamol toxicity [340], which may be attributed, at least partly, to the higher levels of cytochrome P-448 activity in hamster liver [341].

B. Polycyclic Aromatic Hydrocarbons PAH have been used as model carcinogens in numerous studies, involving many different animal species and various routes of administration. In general, genetically “Ah-responsive’’ animals are at increased risk of PAH-induced tumorigenesis, as these chemicals are not only activated by cytochromes P-448 but are also potent inducers of this enzyme system. Following repeated administration of MC intratracheally, responsive mice showed a markedly higher incidence of lung cancers compared with nonresponsive mice [342]. Similarly, responsive mice were more susceptible to MC-induced subcutaneous sarcomas, than were nonresponsive mice [343, 3441. Responsive CH3 mice were more sensitive to skin tumorigenesisfrom dimethylbenz(a)anthracene plus croton oil than were DBA mice [345], and the incidence of benzo(a)pyreneinduced fibrosarcoma was higher in responsive C 3 W e mice than in nonresponsive DBN2 mice [346]. The urinary excretion of mutagenic benzo(a)pyrene intermediateswas higher in responsive mice pretreated with BNF than in similarly treated nonresponsive mice [347]. However, benzo(a)pyrene administered orally to pregnant nonresponsive mice gave rise to more malformations and intrauterine toxicity than did the same administration to



43

responsive mice, possibly because with the nonresponsive animals no cytochrome P-448 induction occurs, benzo(a)pyrene metabolism is not increased, and thus more of the compound reaches the embryos where it exerts its toxicity [348]. However, when benzo(a)pyrene was administered intraperitoneally, a route which avoids first-pass elimination before reaching the embryo, intrauterine toxicity was higher in responsive mice [349]. Destruction of primordial oocytes by 7,12-dimethylbenz(a)anthracene, benzo(a)pyrene, or MC was more rapid in responsive mice, in which these chemicals all induced cytochrome P-448 activity; furthermore, simultaneous administration of the cytochrome P-448 inhibitor a-naphthoflavone inhibited this oocyte toxicity [350]. Nevertheless, some studies have failed to show any correlation between PAH-induced carcinogenesis or toxicity and the presence of the Ah receptor, as indicated by cytochrome P-448 induction.Topical application of benzo(a)pyrene or dimenthylbenz(a)anthracene to mouse skin followed by repeated application of the promoter phorbol ester, in the classical skin cancer model, did not reveal any differences between responsive and nonresponsive mice, and similarly, the toxicity of benzo(a)pyrene following a single intraperitoneal dose showed no correlation with the Ah receptor [351, 3521. However, in these studies the carcinogen was administered as a single dose and it was therefore metabolized/activated by the basal cytochrome P-448 activity, that is, no induction occurred, and although basal levels tend to be higher in the responsive mice, the difference is not marked [136]. In contrast, a single subcutaneous dose of dibenz(a,h)anthracene was much more effective in producing tumors in responsive than in nonresponsive strains of mice; however, in the metabolism of this carcinogen by liver microsomes from MC-treated mice, responsive animals not only showed higher overall metabolism but also directed more of the metabolism toward the formation of the 3,4-diol, the precursor of dibenz(a,h)anthracene-3,4-diol-1,2-epoxide, considered to be the ultimate carcinogen [353]. The importance of dosage regimen is exemplified by studies which showed that a-naphthoflavone, a specific inhibitor of cytochrome P-448, decreased dimethylbenz(a)anthracene-induced skin tumorigenesis only if applied within 12 hours of the carcinogen, emphasizing the importance of activation during the first few hours after carcinogen administration [354]. a-Naphthoflavone administered orally to pregnant rats, followed 1 h later by 7,12-dimethylbenz(a)anthracene (DMBA), resulted in fewer skin papillomas and lung adenomas in the F, generation treated with the promotor 12-0tetradecanoylphorbol-13-acetatefor 12 weeks, than when no flavone was given, possibly because it inhibits the cytochrome-P-448-dependent formation of the carcinogenic diol-epoxides in the dams [355]. The carcinogenic effect of DMBA following topical skin application was inhibited by high doses of anaphthoflavone, presumably by inhibiting the activation of the carcinogen;

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however, the dose response to a-naphthoflavone is not linear, and at some low doses of the flavonoid, the skin tumorigenesis of DMBA was enhanced, indicating that this cytochrome P-448inhibitor is not always anticarcinogenic [356]. Different neoplastic responses are probably mediated by different mechanisms, for induction of leukemia by topical application of MC was seen only in nonresponsive mice, that is, cytochrome-P-448-noninducible, whereas these animals were resistant to skin tumors induced by this carcinogen [357]. TCDD at high doses induces cytochrome P-448activity in nonresponsive as well as responsive mice, and simultaneous subcutaneous administration of TCDD enhanced the carcinogenicity of MC, probably the result of cytochrome P-448induction and increased conversion of MC to its ultimate carcinogen [358]. A number of studies have focused on the teratogenicity of the polycyclic aromatic hydrocarbons rather than on their carcinogenicity; for example, benzo(a)pyrene administered at day 7 or 10 of gestation was more toxic in utero and more teratogenic in responsive than in nonresponsive mice [349].

C. Aromatic Amines and Amides Rat embryos exposed transplacentally to MC, and subsequently cultured in vitro and treated with 2-AAF, exhibited marked increases in malformations compared with embryos from animals exposed transplacentally to PB or vehicle [241]; liver homogenates from MC-pretreated animals only were able to catalyze 2-AAF activation. Earlier studies showed that MC pretreatment of animals protected against 2-AAF-induced tumorigenesis [359], despite the fact that the N-hydroxylation of the latter is catalyzed by cytochromes P-448[ 1311; furthermore, after simultaneous treatment of rats with MC and 2-AAF, despite an increase in the N-hydroxylation of the amide in vitro, the urinary excretion of the N-hydroxy derivative in vivo was significantly decreased [220, 3601. A possible explanation is that the cytochromes P-448more readily catalyze ring hydroxylation of 2-AAF, which leads to detoxification [219]; or alternatively, MC may act as a competitive inhibitor of 2-AAF N-hydroxylation, as both are good substrates of cytochromes P-448[361].

D. Azobenzenes

3'-Methyl-4-dimethylamino-azobenzene(MDAB) is a hepatocarcinogen primarily activated by cytochromes P-448 [226]. In 10 generations of MDAB-exposed Donryu albino rats, carcinogen-resistant descendants were seen in the fourth to eighth generations; the resistant generations had low hepatic AHH activity and low cytochrome P-448induction by MC or PCB,and



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liver microsomal preparations were poor activators of MDAB and other chemical carcinogens to mutagens in the Ames test [362]. In contrast, both resistant and nonresistant rats showed the same response to induction with PB.

E. Polyhalogenated Biphenyls Polyhalogenated biphenyls differ greatly in their chemical toxicity and carcinogenicpotential. Those PCB which have chlorine substituentsin the ortho position(s) of the molecule are nonplanar, are preferentially metabolized by the Phytochromes P-450, and have relatively lower toxicity. In contrast, those PCB which have unsubstituted ortho position(s) and have chlorine substituents in the meta/para positions are planar, are preferentially metabolized by the cytochromes P-448, and interact with the Ah receptor resulting in cytochrome P-448 induction and high potential toxicity/carcinogenicity [37, 3031. Exposure to polybrominated biphenyls (PBB) has been associated with a number of toxic effects [363] although human epidemiological studies have proved largely inconclusive [364,365]. PBB are sold commercially as complex mixtures and, like their PCB analogues, are mixed-type inducers; that is, they induce both PB-cytochromes P-450 and cytochromes P-448 [37]. It is interesting that congeners which induce the PB-cytochromes P-450 are relatively nontoxic compared with the mixture Firemaster, which induces the cytochromes P-448, indicating that the cytochromes P-448 may largely be responsible for the toxicity of these chemicals [366,367]. The immunotoxicity of polyhalogenated biphenyls is also mediated through the Ah receptor of lymphoid tissue [275,368], and causes thymic atrophy and suppression of the antibody response in responsive but not in nonresponsive mice. The toxicity of TCDD, another polyhalogenated polycyclic compound, was markedly higher in responsive mice than in nonresponsive strains [369]. Taken as a whole, these studies indicate the importance of the Ah locus in mediating the toxicitykarcinogenicity of exogenous chemicals.

F. Multidrug Resistance Human tumors can develop resistance to anticancer drugs, and similarly, rats and other mammals, especially with liver tumors, are able to acquire resistance to carcinogens and toxic chemicals [370]. This acquired resistance to chemical toxicity, known as “multidrug resistance” (MDR) is associated with a 170,000-dalton membrane glycoprotein(s) (P-glycoprotein) which binds xenobiotics, and which is concerned as an energy-dependent efflux pump in their transport from the cell, resulting in lower intracellular concentrations of the chemicals. Levels of mRNA for the MDR gene are elevated in preneoplastic


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and neoplastic liver lesions, by partial hepatectomy, and by treatment of animals with aflatoxin B,, 2-AAF, or N-hydroxy-ZAAF, but not 7-hydroxy-2-AAF. Treatment of rats with TCDD or isosafrole, which induces cytochrome P450 I, simultaneously increases the expression of the MDR gene. From similar studies in responsive (B6) and nonresponsive mice (D2), it appears that the Ah locus is involved in the gene expression of MDR, since MDR is induced by aflatoxin B,, AAF, and TCDD in the B6, but not the D2, mouse. The coregulated expression of MDR and the cytochrome P450 I gene family, and possibly also other cytochrome P-450 supergene families, may constitute a physiological mechanism for the protection of the organism against the adverse effects of natural and synthetic xenobiotics [370, 3711.

VIII. FACTORS MODULATING CYTOCHROME P-448 ACTIVITY A. Tissues

Because of their low levels of mixed-function oxidase activity, extrahepatic tissues have received relatively less attention than liver, and cytochrome P-450 proteins have been purified only from liver and lung and, in the last few years, from the gastrointestinal tract. Quantification of the various cytochrome P-450 isozymes has been achieved largely by monitoring the metabolism of specific substrates or by the use of polyclonal and monoclonal antibodies prepared against the individual purified cytochromes.

1. Lung Using the EROD assay, cytochrome P-448 activity was lower in the lung than in the liver of rats [14], and in some cases was not even detectable [104]. Low levels of EROD activity were also detectable in rabbit lung [41]. The lower activity in lung compared to liver was confirmed by radioimmunoassay with monoclonal antibodies against cytochrome P-45Oc [372]. Similarly, in rabbit lung, immunofluorescenttechniques detected LM2only, and LM, and LM, were not detectable [373]. Using the more sensitive Western blotting technique, pulmonary levels of LM, were found to be c 3% of the total microsomal cytochromes P-450 [374]; LM, was still undetectable. The tissue distribution of LM, differs significantly from that of LM2 and LM, in that it is found in pneumocytes other than the Clara cell, the type I1 cell, and alveolar macrophages [375]. Species differences occur in the induction of cytochrome P-448 activity in the lung. In rat lung, cytochrome P-448 (EROD activity) is induced only by MC



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and 7,12-dimethylbenz(a)anthracene7 and to a lesser extent by P-NF [103]. MC treatment of rats induced proteins in the lungs which were antigenically similar to rat liver cytochrome P-45Oc, the concentrations being much lower than in liver, as indicated also by the EROD assay [ 104,370,376,3771. In MC-induced rats, cytochrome P-45Oc was localized in Clara cells, alveolar type I1 cells, and endothelial cells; in contrast, the endothelial cells were devoid of cytochrome P-450b in PB-treated rats [378]. A major form of pulmonary cytochrome P-450, different from the hepatic proteins, has been purified from MCpretreated rats [379]; this form readily catalyzed the oxidation of benzo(a)pyrene to various phenols, diols, and quinones [380]. However, cytochrome P-450d appears not to be induced in the lung, at least not by the potent inducing agent TCDD [158]. Similarly, MC pretreatment of rabbits increased the pulmonary level of total hemoprotein, accompanied by a shift of the CO difference absorption maxima to lower wavelengths and an increase in AHH activity [381]. The extent of induction in rabbit lung was low compared with that seen in liver. Pulmonary EROD activity was also induced in rabbit by TCDD and by the PCB mixture, Aroclor 1260 [41]; TCDD induced LM, and LM, [41, 3731, although other workers observed induction of LM, only [382]. Treatment of rabbits with TCDD markedly increased LM, in Clara and type I1 cells, and even more in macrophages, in which no basal activity was detectable [375]; in all cases the increases in concentration of this isozyme were accompanied by similar changes in EROD activity. Similarly, in mouse lung, cytochrome P,-450 but not P,-450 was induced by MC [155]. In contrast, in untreated mice, cytochrome P,-450 activity was higher than cytochrome PI-450 activity. The inducibility of cytochromes P-448 in mouse lung was studied by the use of a monoclonal antibody raised against the rat hepatic cytochrome, which cross-reacted with the murine pulmonary cytochrome P-448 [1761; in MC-induced mice, cytochrome P-448 was present in lung and hepatic parenchymal cells, but this isoenzyme was not detected in untreated or PB-induced lungs. In hamster, treatment with MC, P-NF, or Aroclor 1254 also stimulated AHH activity in lung, whereas hepatic levels were not significantly affected [383, 3841. This sensitivity of the lung to induction by MC may partly explain why the hamster is so susceptible to tumors of the respiratory tract [385]. In contrast to the cytochromes P-448, the PB-cytochromes P-450 of liver appear to be the major constitutive forms present in lung of rat, mouse, and rabbit. Cytochrome P-450b, but not cytochrome P-450e, was present in the lungs of male and female rats, together with other polypeptides, most of which were related to cytochrome P-450b [378, 386, 3871. In rabbit lung, LM, and LM, were present, but not LM,, and LM, was detected only in trace amounts [373, 3741. However, in contrast to liver, the PB-cytochrome P-450 isozymes of lung


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are not induced by PB[176, 373, 388, 3891, although a small but significant increase in cytochrome P-450b in rat pulmonary microsomes was demonstrated by Western blotting [378]. The alcohol inducible P-45Oj is barely detectable in the lung but it may be induced by appropriate agents [35]. The lung is a major site of prostaglandin metabolism, which is induced by progesterone. These substrates are not metabolized by cytochromes P-448, but by other forms of the hemoprotein, two of which have been isolated from lungs of rabbits treated with progesterone; one of these (P-45Op-1) catalyzed the metabolism of exogenous chemicals while the other (P-45Op-2) catalyzed the w-hydroxylation of prostaglandins and the w- and (o-1)-hydroxylations of palmitate and myristate [390]. Lung microsomes from pregnant rabbits are also very active in the w-hydroxylation of prostaglandins [391, 3921. In conclusion, the lung contains very little cytochrome P-448 activity, but one of the associated proteins, cytochrome P-450c, can be modestly enhanced by potent inducing agents, while cytochrome P-450d appears not to be readily inducible [382]. However, the lung contains other cytochrome P-450 proteins which are active in the metabolism of endogenous substrates. 2. Kidney

In MC-treated rats, cytochromes P-450 antigenically related to hepatic cytochromes P-448 were detected in kidney but at levels markedly lower than those seen in liver [372, 3761. Using monoclonal antibodies recognizing MC-induced cytochromes, only small amounts with this epitope were detected in kidney [393]. From a comparison of warfarin metabolism, the MC-induced kidney cytochrome P-450 was similar to the hepatic hemoprotein [394]; by the EROD assay untreated kidney was shown to have very low cytochrome P-448 activity but this was induced by PAH, P-NF, and 2-AAF [ 14, 1041. With TCDD as inducing agent, immunoquantification showed a marked increase in cytochrome P-450c, which accounted for 30% of total rat kidney cytochrome P-450 [158]. In the untreated rabbit, LM, was detected in the proximal kidney tubule; LM4 and Lm, were not present but appeared following induction with TCDD [373]. An MC-inducible low-spin form of cytochrome P-448 catalyzing the hydroxylation of benzo(a)pyrene and resembling LM, has been isolated from the cortex of rabbit kidneys [395]. Similarly, in mouse kidney, cytochrome P,-450 but not P3-450 was induced by MC [155]. Neither P-450b nor P-450e mRNA were detected in the untreated, PB- or MC-treated kidney [387]. However, cytochrome P-452 was present in the untreated kidney and was induced by clofibrate [63]. Similarly, the alcoholinducible cytochrome P-45Oj is also present in the kidney and may be induced by agents such as alcohol and isoniazid [35, 3961.


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3. Gastrointestinal Tract EROD activity was detectable in intestinal microsomes from untreated rats and was induced by P-NF [397]. After TCDD administration to rats, cytochrome P-450c, determined by immunoquantification with a monoclonal antibody, accounted for 70% of the total intestinal hemoprotein content [ 1581. Two forms of cytochrome P-450 active in benzo(a)pyrene hydroxylation were separated from the intestinal microsomes of rabbits treated with MC [398]. Subsequently, three forms of the cytochrome were isolated from colon mucosal microsomes of untreated rabbits [399]: One of these forms, namely, cytochrome P-448c, hydroxylated benzo(a)pyrene and also demethylated benzphetamine and aminopyrine, and (despite its nomenclature) is unlikely to be a member of the cytochrome P-448 gene family; a second form, cytochrome P-45OC,catalyzed the w-hydroxylation of prostaglandins and the w- and (w-1)-hydroxylations of myristate and laurate; the third form, cytochrome P-450,, had no detectable activity toward prostaglandins or fatty acids but demethylated aminopyrine and benzphetamine [399]. The second isoenzyme, cytochrome P-450,, is catalytically similar to the protein isolated from lungs of rabbits treated with progesterone [390] and also from microsomes of the small intestine [395, 3991 and kidney cortex of rabbits treated with PB and MC [400]. In mouse intestines, cytochrome P,-450 was induced by MC, and cytochrome P,-450 was not induced, although its constitutive activity was relatively high [155].

4. Other Tissues Cytochrome P-450 proteins and activity have been detected in most tissues. Cytochromes LM2and LM, were detected immunologically in rabbit aorta, and both LM, and EROD activity were induced in this tissue by TCDD [401]. Using immunohistochemical techniques, cytochrome P-45Oc has been detected in rat aorta, as well as other tissues such as heart, spleen, brain, and testes [158,225]. The relevance of these findings in the metabolism of endogenous and exogenous substrates remains unclear, but the biosynthesis and deactivation of steroid hormones and eicosanoids must be major functions. In the bladder mucosa of rabbits, LM, and LM,, but not LM4,were detected using immunochemical techniques [402]; significant EROD activity was also detectable, but none of the cytochromes P-450 were inducible by pretreatment with PB or TCDD. Cytochrome P-45Oc has been demonstrated immunohistochemically, and by determining mRNA, in the ventral prostate of rats pretreated with P-NF, but not in PB-treated or untreated animals [156,403, 4041; only P-45Oc is induced, in


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contrast to the liver in which tissue both cytochrome P-448 enzymes are increased [405]. Microsomal preparations from induced rat ventral prostate converted benzo(a)pyrene to the 7,8-dihydrodiol, and activated a variety of other carcinogens to mutagenic products in the Ames test [406]; cytochrome P-450b was not present in these preparations. In contrast to these animal studies, a cytochrome P-450 protein immunologically related to cytochrome P-450b has been detected in human prostate [407]. Mixed-function oxidase activity has been detected in the nasal mucosa of rabbits, and using specific antibodies to rabbit liver cytochromes, LM, was shown to be the major isozyme, with LM,, but not LM,, also present. No induction of these proteins was observed following PB treatment [408]. The alcohol-inducible cytochrome P-45Oj has also been detected in nasal mucosa [3961. Placentas from various species appear to contain no PB-cytochromes P-450 activity and show no induction of these enzymes with PB or other drugs; in contrast, they do exhibit low AHH and EROD activities which can be markedly induced by PAH or exposure to tobacco smoke [ 157,4091. Similarly, cigarette smoking stimulated human placental AHH and EROD activities [410-412], and placentas from Chinese women exposed to rice oils contaminated with PCB exhibited higher levels of AHH and EROD activity than control subjects [308, 4131; these activities correlated with a protein which cross-reacted with an antibody to LM,. Moreover, the smoking-induced AHH activity in human placenta was inhibited by a monoclonal antibody to a cytochrome P-450 from the liver of PAH-induced rat [414]. Furthermore, a partially purified cytochrome P-450 from the placentas of smokers was shown to catalyze the cytochrome-P-448-dependent metabolism of benzo(a)pyrene and 7-ethoxyresorufin, but a similar preparation from nonsmokers did not [415, 4161. In another study, placental microsomes from nonsmoking women did not metabolize benzo(a)pyrene, but midgestational and full-term placental preparations from smokers formed phenols, and all three diols [417]. Cytochromes P-450 were undetectable in mammary microsomes from virgin rats, but following pretreatment with PAH, enzyme activity was detected [418]. Exposure of human mammary epithelial cells to benz(a)anthraceneresulted in induction of the low-spin form of cytochrome P-448 [306,307]. Human breast tumor cell lines have been employed as activators of PAH [416] and both stromal and parenchymal cells could activate the precarcinogen 7,12-dimethylbenz(a)anthracene, for which breast is a target tissue [419]. Both types of cells could convert benzo(a)pyrene to the 9,lO- and 7,8-diols when these cells were exposed to TCDD; induction of P-45Oc was observed, but surprisingly no change was seen in the metabolism of 7,8-diol of benzo(a)pyrene to its diol-epoxide [420]. In summary, cytochrome P-448 levels are generally low in extrahepatic



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tissues but cytochrome P-45Oc may be markedly increased by potent inducers such as TCDD, P-NF, cigarette smoke, and PAH [382]. For example, in rat prostate, constitutive EROD activity is just detectable, but after treatment with P-NF a 7500-fold induction has been reported [403]. In contrast, cytochrome P-450d appears not to be expressed even after exposure of the animals to inducing agents [155, 158, 382, 4131.

B. Sex Numerous studies from the 60s have established that, both in vivo and in virro, the male rat metabolizes a number of substrates more rapidly than does the female; in these studies no discrimination was made between the activities of the different cytochromes P-450 [421, 4221. In contrast to these studies, and using EROD to determine cytochrome P-448 activity, it was shown that with rat [423,424], and with both “responsive”and “nonresponsive”mice [1361, the female exhibited higher cytochrome P-448 activity, thus indicating that cytochromes P-448 and Pkytochromes P-450 have different sexual distributions. In confirmationof this, studies in which cytochrome P-450 mRNAs were quantified by translation and immunoprecipitation, those coding for cytochrome P-450b were higher in males than in females, while in contrast, mRNAs coding for cytochrome P-45Oc peptides were twice as high in females compared to males [66]. As microsomal cytochrome P-450 levels are regulated by mRNA levels [425, 4261, it can be concluded that the known sex differences in rates of xenobiotic metabolism may simply reflect a different distribution of the various cytochrome P-450 isozymes in the two sexes. The higher cytochrome P-448 activity in female rat may account for the higher conversion of paracetamol to its reactive intermediates by the female than by the male rat, which also excretes more of the drug as mercapturate conjugates [427], and is more susceptible than the male rat to paracetamol toxicity [428]. The higher cytochrome P-450 activity in the male rat becomes evident at 30 days after birth. If male rats are castrated soon after birth, this sex difference is abolished, and this phenomenon whereby neonatal testosterone levels determine enzymic activity in later life, is known as “neonatal imprinting” [429]. Similarly, imprinting occurs when PB is administered during the neonatal period [430-433], but no such effect is observed when MC is used as inducing agent, indicating that cytochromes P-448 do not undergo neonatal imprinting [434]. Differences in the metabolism of endogenous chemicals by the two sexes can also be accounted for by the levels of other cytochromes P-450. For example, the 16a-hydroxylation of testosterone is 10-fold higher in male than in female 129/J mice and this is due entirely to the levels of cytochrome P-450,, [ll]; similarly, the 15a-hydroxylation of testosterone [lo] as well as the 15P-


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hydroxylase of androstenediol disulfate are female specific [ 131.The expression of these hydroxylases appears to be irreversibly determined by neonatal imprinting [429, 4351; testicular androgens imprint the ability to express male-specific cytochromes P-450 and to suppress female-specific forms, with sexual differentiation being regulated by pituitary growth hormone [436]. No sex differences were apparent in P-45Oj and the related activities [61].

C. Species Differences As the cytochromes P-450 exhibit different substrate specificities, and stereoand regio-selectivities, the biological fate of a chemical depends not only on the total levels of the cytochromes P-450, but also on the relative abundance of the various isoenzymes. The nature of the cytochrome P-450 population will therefore determine if a chemical will be detoxicated and excreted, or will be activated to interact covalently with cellular components, thus giving rise to toxicity, mutations, and malignancy. The well-known differences in the susceptibility of different animal species to chemical toxicity may simply represent the presence of different cytochrome P-450 isozymes. An appreciation of the substrate specificities of the component cytochromes P-450, and their relative abundance in laboratory animals and man, will no doubt make extrapolation of animal toxicity data to humans a more precise exercise.

1. Structural Aspects Mouse, rat, and rabbit are the three most extensively studied animal species with respect to the mixed-function oxidase system. Very similar forms of the cytochromes P-450 are induced in different species by the various inducing agents. A comparison of the partial cDNA sequence of rat cytochromes P-45Oc and P-450d with MC-inducible mouse cytochromes P,-450 and P,-450 revealed high homology between P-45Oc and P,-450 DNA, and between P-450d and P,-450 [ 191. Furthermore, rabbit LM, mRNA is orthologous to rat P-450d [82] and mouse P,-450 [19]. Rat cytochrome P-450b and rabbit LM, have 80% similarity in their primary structures [437], and amino acid and DNA sequence analysis revealed homology between the rat cytochromes P-450b, P-450e, and rabbit LM, [59, 73, 4384401. Finally, P-45Oj in the rat is orthologous to the rabbit LM,, [440] and hamster proteins [441].

2. Immunological Aspects The advent of monoclonal antibodies has allowed the rapid identification and quantification of antigenically similar cytochrome P-450 proteins, such as



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cytochromes belonging to the same gene family. Thomas et al. [110] have raised nine monoclonal antibodies against cytochrome P-450c, reacting with six distinct epitopes, one of which was shared with cytochrome P-450d. The same workers, using monospecific polyclonal antibodies, detected cytochrome P-45Oc at low levels in the liver of rats and at relatively higher levels in rabbit and guinea pig livers, whereas no protein was detectable in the mouse and hamster; a protein related to cytochrome P-450d was present in all animals [110]. EROD activity was significantly higher in hamster than in rat [341], but as EROD activity measures mostly cytochrome P-450c, and to a much lesser extent cytochrome P-450d, it cannot be decided which cytochrome P-448 isoenzyme is responsible for the high activity. The possibility that EROD activity and the monoclonal antibodies determine different but immunologically related proteins cannot be excluded. Using the same monoclonal antibodies, both cytochrome P-448 isozymes were induced by MC in rat, hamster, guinea pig, rabbit, and “responsive” mice, but not in “nonresponsive” mice [110]. Similarly, isosafrole induced cytochromeP-450d in all animals, but cytochrome P-45Oc in rat, rabbit, and hamster only.

3. Extent of Induction Species differences also occur in the extent of induction; MC induced cytochrome P-45Oc markedly in rat and responsive mice, and to a less extent in hamster, rat, and guinea pig. Using two monoclonals against cytochrome P-450c, similar findings have been reported by Cheng et. al. [376]. When EROD activity was determined, induction was much higher in rat than hamster [103]. Cytochrome P-450d was most extensively induced in the responsive mouse, followed by rat, guinea pig, hamster, rabbit, and nonresponsive mouse [110]. Induction of cytochrome P-45Oc by isosafrole was highest in rat, followed by guinea pig. A marked species difference is seen in the induction of P-45Oj which in the rabbit, but not in the rat, is induced by imidazole [442].

4. Substrate Criteria A similar comparison of hepatic cytochrome P-448 activity-using the EROD assay and undertaken with various inducing agents, including MC, 2-aminoanthracene, and Aroclor 1254-showed that hepatic cytochrome P-448 activity is poorly induced in hamster, but markedly in the rat; a single i.p. administrationof Aroclor 1254 caused a 150-fold increase in EROD activity in rat liver but only a 3-fold increase in hamster [104,341]. Similarly, liver AHH activity was not induced in the hamster by MC [443]. These findings indicate


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that the cytochrome P-448 isozymes induced in hamster share common epitopes with the rat proteins but have different affinities toward certain substrates, such as 7-ethoxyresorufin and benzo(a)pyrene. This is further supported by the fact that Aroclor 1254-induced liver preparations from rat and hamster activated benzo(a)pyrene to the same extent, despite the fact that EROD activity in the former was 14 times higher [341]; two partially purified cytochrome P-448 proteins from hamster showed lower AHH activity than the orthologous rat proteins [444]. The guinea pig, similar to hamster, is relatively refractory to AHH induction by MC [443,445,446]; TCDD treatment produced a 500-fold increase in EROD activity in rat hepatocytes but only a 6-fold increase in the guinea pig [447]; moreover, TCDD induced its own cytochrome-P-448mediated metabolism in rat but not in guinea pig. The poor response of the guinea pig to cytochrome P-448 inducers may be, at leas?partly, responsible for its well-known resistance to 2-AAF hepatocarcinogenesis,since the activating N-hydroxylation is catalyzed by cytochromes P-448 [131], which in the rat is self-induced by 2-AAF [ 1811. Studies using warfarin as a substrate probe coupled with immunoinhibition and immunoelectrophoresishave indicated that cytochrome P-450 isozymes in P-NF-induced “responsive” B, mice are different from rat cytochrome P-450c, while cytochromes induced by PB or PCN are the same in both species [448]. These observations indicate that p-NF may induce different cytochrome P-450 proteins in mouse and rat; indeed the substrate specificities of cytochromes P-448 in mice, rats, hamsters, guinea pigs, and rabbits indicate that hepatic cytochrome P-448 comprises several enzyme proteins, which differ in different animal species and differ in the substrates which they oxygenate [443]. Cytochromes P-450 are also present in the livers of fish, and most of the isozymes isolated and characterized appear to be of the cytochrome P-448 type [449, 4501.

D. Strain Differences Relatively little attention has been devoted to the study of strain differences in the cytochrome P-450 proteins. The cytochrome P-45Oc proteins from Holtzman and Long-Evans rats are markedly similar, having identical spectral, immunochemical, and catalytic activities [451]; similarly, cytochrome P-450b proteins from these two strains displayed similar substrate specificities and spectral and immunochemical properties [451]. These immunologically related cytochrome P-450b proteins appear to be products of distinct mRNAs [46]. The cytochrome P-450a (P450 I1 A) proteins of Long-Evans and Sprague-Dawley rats are similarly immunologically related (4521.



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Strain differences in mice have already been discussed in reference to the Ah receptor.

E. Age In all animal species including man, it has been established, using a limited number of “standard” mixed-function oxidase substrates,that activities are very low at birth but increase rapidly to adult levels [453,454] and then subsequently decline in geriatric animals [455]. Even in these initial studies it was observed that not all substrates followed the same developmental pattern, indicating that cytochrome P-450 isozymes may not develop coordinately; for example, the cytochrome-P-448-mediated 2-hydroxylation of biphenyl was present in neonatal rat and rabbit livers but was undetectable during adulthood [456,457]. These observations have been confirmed recently using the more specific EROD assay, showing that cytochrome P-448 activity is relatively high in the neonatal rat, reaching a maximum 2 weeks after birth, and then decreasing with age. In contrast, the PB-cytochromes-P-450-mediated N-demethylation of benzphetamine was low at birth and increased with age [458]. These observations were further confirmed by Blanck et al. [424], who showed that 3-week-old animals exhibited markedly highly EROD activity than 8-week-old animals. It is interesting to note that the ontogeny of cytochromes P-448 is closely related to that of the cytosolic receptor [459]; in rat liver and lungs the concentration of cytosolic receptor rose rapidly after birth, reaching maximum levels at 1-3 weeks, and then declined with age; at all times the receptor avidly bound TCDD. It is thus possible that basal levels of cytochrome P-448activity may be related to the receptor concentration. A similar ontogenic profile was seen in other species [460]. Similar to cytochromes P-448, the alcohol-inducible cytochrome P-45Oj appears to be higher at around 3 weeks after birth, constituting a quarter of the total cytochrome P-450 content, and then declines rapidly [35]. In other studies, employing Western blotting analyses, this protein was undetectable in the newborn rat but increased rapidly within a week and remained at this level for at least 12 weeks (61). In contrast, the same workers have shown that P-450b was detectable in the neonate and increased with age, reaching a maximum at 4 weeks of age. The higher cytochrome P-448 activity exhibited by the neonate would predispose it to the toxicity of chemicals whose activation is mediated by these isoenzymes. Indeed, neonatal rats convert paracetamol to its reactive intermediates more readily than do adults, and neonatal rats excrete more paracetamol as the mercapturate conjugate [427].


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The extent of induction of cytochrome P-450 proteins by various inducing agents also appears to be age dependent and differs between cytochromes P-448 and PI3-cytochromes P-450. Generally, the inducibility of cytochrome P-450 activities by PB is higher in the young animal and decreases with age [455,456, 4611, whereas induction of cytochromes P-448, as exemplified by the biphenyl 2-hydroxylation and EROD assays, is low in the neonate and increases with age [458]. Similar observations have been made using oligomer probing techniques for mRNA (4621; the extent of induction of nitroanisole 0-demethylase by BNF was higher in 2-year-old rats than in 3- to 5-month animals, whereas the extent of induction of the same activity by PB was less in the older animals [461]. EROD activity is also detectable in the rat fetus and can be markedly induced by transplacental administration of P-NF [ 1571; this increase was due to cytochrome P-45Oc and not to cytochrome P-450d. In contrast, neither P-45Oc nor P-45Od mRNA were detectable in rat fetus after treatment of the dams with MC [462], although other workers reported an increase in murine fetal P-45Oc mRNA only [463]. However, in the rat, P-450b and P-450e mRNAs could not be transplacentally induced before the 21st day of gestation [462].

F. Nutrition Nutrition and diet may modulate the levels of cytochrome P-450 isoenzymes, resulting in changes in the metabolic fate of xenobiotics [464]. The dietmediated effects may be due to chemicals inherent in the diet, to contaminants or food additives, or to compounds generated during the process of cooking. Some types of food have high contents of natural xenobiotics which are potent inducers of the mixed-function oxidase system, such as safrole, flavones, and indoles. Rats maintained on a diet containing cruciferous vegetables, such as sprouts and cabbage, probably because af their high content of indoles, exhibited both high levels of intestinal mixed-function oxidase activity [465] and an altered response to chemical carcinogens [466,467]; similar effects were observed in humans maintained on such diets [468]. Indeed, indole-3acetonitrile, indole-3-carbin01, and 3,3'-di-indolylmethanes,present in cmciferous vegetables, have been identified as naturally occurring inducers of hepatic and intestinal AHH, and of other enzyme activities [469, 4701; these compounds selectively induced the cytochromes P-448 [471]. Liver and intestinal EROD activities have been induced in rats by feeding cabbagecontaining diets [472]; indole-3-carbinol was a potent inducer of liver activity and to a lesser extent of intestinal activity; other indoles also induced liver activity, albeit weakly, while ascorbigen (a product of indole-3-carbinol and ascorbic acid) stimulated only intestinal activity.



57

Rats maintained on these indole-supplemented diets showed protection against PAH-induced carcinogenicity [467], possibly because the indoles may act as competitive substrates preventing activation of the carcinogen, or they may be even more potent inducers of the phase I1 detoxication pathways. Pretreatment of animals with indole-3-carbinol decreased the covalent binding of benzo(a)pyrene metabolites to DNA and protein [471], which accords with both hypotheses. However, as there was no change in the oxidation of benzo(a)pyrene on these diets, and as the formation of the ultimate carcinogen, benzo(a)pyrene-9,10-dihydrodiol [473] was unaffected, competitive inhibition of oxidative activation seems unlikely; the observed stimulation of hepatic glutathione S-transferase activities by these brassica-supplemented diets [473] makes an increase in the detoxication pathways a more likely mechanism of the protective effect against chemical carcinogenesis. Inducing agents also may be generated during the process of cooking, and charcoal-broilingbeef, because of the high levels of PAH,stimulates placental and intestinal mixed-function oxidase activities of rat [474] and humans [475, 4761. A number of aza-arenes have been isolated from food subjected to high cooking temperatures; many of these are activated to mutagens and carcinogens by the cytochromes P-448, and one of these, 2-amino-3-methylimidazo[4,5flquinoline (IQ), has recently been shown to selectively induce cytochrome P-448 activity and its own activation [96,477a]. Other food mutagens such as the pyrolysate products Trp-P-1 and Trp-P-2 also induce cytochrome P-448 activity [95].

IX. HUMAN CYTOCHROMES P-448 Cytochrome P-450 concentrations in human liver are generally lower than those of laboratory animals, and the metabolic rates for many typical substrates are correspondingly slow. However, it is now becoming apparent that isoenzymes of the cytochromes may be encountered in human liver at levels similar to, or higher than, those occurring in laboratory animals. For example, coumarin 7-hydroxylase activity in human liver biopsy samples is particularly high [477b]; N-hydroxylase activity toward the carcinogen 2-AAF is high [478] and the aromatic amide is more readily converted into mutagens by human liver than by rat preparations [479]. Human liver S9 preparations are at least as active as those derived from rat liver in activating aromatic amines such as 4-aminobiphenyl, 2-aminoanthracene, and 2-aminofluorene, and nitrosamines such as nitrosopyrrolidine and nitrosopiperidine to mutagens [479, 4801, but similar to laboratory animals, cannot readily activate polycyclic aromatic hydrocarbons [481]. A number of human hepatic cytochromes P-450 have been isolated and characterized. These do not correspond to the MC-and PB-inducible forms of


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rat liver but, by the use of polyclonal and monoclonal antibodies and cDNA techniques, immunologically related forms were detected in human and animal livers; in human liver a family a two isozymes, immunochemically and structurally related to cytochromes P-448, have been demonstrated. Antibodies against cytochromes P-45Oc and P-450d interacted in Western blots with 9 out of 31 human samples, showing marked interindividual variation [482], as has previously been reported for human mixed-function oxidase activities in uitro [483]. Immunoblot analysis on 14 human hepatic microsomal preparations revealed the presence of a band reacting with cytochrome P-450d, with more than a 10-fold variation in concentration [160]; only one of the specimens contained, in addition, a band that reacted with cytochrome P-45Oc. Polyclonal antibodies to rabbit LM, and LM, were used to probe human hepatic microsomes from 6 subjects [484]; both antibodies recognized a corresponding protein in Western blots but no correlation could be obtained between the concentrationsof these forms and a number of mixed-function oxidase activities including AHH and 2-AAF N-hydroxylase. Antibodies against P-450d were very effective in inhibiting the activation of 2-aminofluorene,IQ, its methylated analogue MeIQ, and the related quinoxaline MeIQx, by human microsomes [485); in contrast, antibodies to P-450b were ineffective. Studies employing inhibitors of various cytochrome P-450 proteins revealed that human hepatic EROD activity resembles the induced cytochrome P-448 activity seen in the rat [4861. Two human cytochromes P-45O-denoted hp P-450, and hp P-4506, and correspondingto rabbit LM, and LM,-have been cloned from a human hepatic cDNA library [487]. Analysis of the DNA sequence showed that hp P-450, shares 83% and 75% homology with LM, and LM, mRNAs, respectively; and hp P-4506 shares 79% and 72% homology with LM6 and LM,, respectively. Furthermore, cloned hp P-450, preferentially hybridizes with LM, and murine P,-450 mRNAs, whereas hp P-4506 preferentially hybridizes with LM, and murine P,-450 mRNAs [487]. The cDNA nucleotide sequences of human and mouse cytochromes P-448 had 63% homology,while the amino acid sequences displayed 80% homology. Homologies between the human cytochrome P-448 protein, mouse cytochrome P,-450, and rat cytochromes P-450c, P-450d, and P-450e were 68%, 79%, 68%, and 35%, respectively. However, the same workers suggested that man has only one cytochrome P-448 isoenzyme (P,-450), in contrast to other animal species studies [488]. These observations provide overwhelming evidence of the existence in human liver of gene(s) which are related to cytochromes P-448. Much less information exists on the induction of human cytochromes P-450, and this is limited largely to a comparison of smokers versus nonsmokers. The levels of EROD activity are generally lower in human liver than in rat [482], but activity may be significantly increased in the livers of smokers (486,4891;



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a monoclonal antibody against MC-induced rat liver microsomes inhibited this induced activity, but this was not inhibited by a monoclonal against PB-induced liver microsomes. Furthermore, AHH activity in lymphocytes was higher in smokers than in nonsmokers [490]. The only other human tissue studied extensively is the placenta, in which cytochrome P-448 activity has been detected using the EROD assay and the regio- and stereo-selective metabolism of warfarin [491]. The lack of detection of pentoxyresorufin dealkylase activity indicates the absence of PBcytochromes P-450. As with the liver, smoking, both active and passive, induces placental EROD activity [492], and a cytochrome P-450 isozyme partially purified from placentae of smokers catalyzed the metabolism of 7-ethoxyresorufin and benzo(a)pyrene, whereas a similar preparation from nonsmokers did not [415,416,493]. In the placenta from smokers, EROD activity was inhibited by a monoclonal to cytochrome P-448 but not by one specific to P R cytochrome P-450 [492]. Chinese women accidentally exposed to PCB exhibited higher placental AHH and EROD activities than control subjects [308, 4131. The increased enzyme activity was associated with increases in a protein immunorelated to LM, [413]. Genetic Polymorphism. Human genetic polymorphism in xenobiotic hydroxylations was first shown to occur with the drug debrisoquine, and was subsequently found to involve many other drugs and several families of the cytochromes P-450 [494,495]. Slow metabolizers appear to have absent or very low levels of the specific cytochromes P-450 which catalyze the hydroxylations of the various drugs and other xenobiotics [496]. The intraindividual differences seen in the responses of humans exposed to tobacco smoke might indicate a similar polymorphism in the activities of the cytochromes P-448, but at present insufficient evidence is available. A significant correlation has been described between the hydroxylation of debrisoquine and EROD activity in human microsomes [497], and debrisoquine has been shown to competitively inhibit EROD activity; however, inducers of cytochrome P-448 activity had no effect on debrisoquine hydroxylation [498]. Slow and fast metabolizers of the carcinogen 2-AAF have been identified among 28 samples of human liver [499]. Although in most cases fast activators (high N-hydroxylation) were also fast detoxicators (high ring hydroxylation), different phenotypic patterns were discerned indicating that a fast activator was not necessarily a fast detoxicator. As only the cytochromes P-448 catalyze the N-hydroxylation of 2-AAF [ 1311, these results provide indirect evidence that the cytochromes P-448 may also exhibit genetic polymorphism. The Ah levels in human tissues may also be under genetic control or modified by the chemical environment, so that high levels might predispose an individual to chemically induced malignancy, for it is interesting to note that the Ah receptor was detected in only 10 out of 53 human lung samples [301].


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Good correlation has also been shown to exist between the inducibility of AHH activity and the incidence of pulmonary or oral squamous cell carcinoma in humans [50-502]. No similar relationship has been observed with other types of cancer [503,5041. The EROD assay may provide a more specific parameter than AHH activity for establishing correlationsbetween cytochrome P-448 tissue concentrations and the susceptibility of human individuals to cancer.

X. COMPETITION BETWEEN CYTOCHROMES P-448 AND PB-CYTOCHROMES P-450 Structural models of hepatic microsomal mixed-function oxidation envisage a central molecule of the cytochrome P-450 reductase donating electrons to 6-8 surrounding cytochrome P-450 proteins [505]. Such an arrangement raises the possibility of competition between the various cytochrome P-450 proteins for reducing equivalents and may explain why the membrane-bound enzymes in microsomal preparations are less active than the purified enzymes in reconstituted systems. The 2-hydroxylation of biphenyl catalyzed by cytochromes P-448 in a reconstituted system with limited amounts of reductase, was inhibited by PB-cytochromes P-450, although the latter only supported the 4-hydroxylation of this substrate; in contrast, cytochromes P-448 did not inhibit the PB-cytochrome P-450-inhibited N-demethylation of benzphetamine [506]. The inhibitory effect of the hemoproteins could be reversed by addition of more reductase [506]. These experiments indicate that Pkytochrome P-450 proteins have a higher affinity for the reductase than do the cytochromes P-448. A similar interaction has been reported using the metabolism of benzo(a)pyrene as a probe [507], which again indicates competition for the reductase; at low levels of cytochromes P-448, addition of PB-cytochromes P-450 increased the metabolism of benzo(a)pyrene, whereas at higher concentrationsof cytochrome P-448, activity was inhibited by addition of PB-cytochrome P-450. In studies with eight purified rat liver cytochromes P-450, using warfarin as substrate, each isoenzyme inhibited the metabolic capacities of the others to variable extents [508]; these findings were not due to limiting reductase and were attributed to enzyme aggregation. However, when testosterone was used as the substrate to monitor the various isoenzymes, no loss of activity was observed when purified cytochromes P-450a, P-450b, and P-45Oc were reconstituted in binary or ternary mixtures of the cytochromes [509]; the lower rates of metabolism observed in microsomes were considered to be due to limiting levels of the NADPH-cytochrome P-450 reductase, which cannot therefore support maximum metabolic rates.


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XI. CONCLUSIONS Recent cDNA studies of the individual members of the superfamily of cytochromes P-450 have indicated that they are ubiquitous in all living organisms and tissues, and have evolved over millions of years from one or more primitive ancestral forms into the vast array of isoenzymes that characterize the different modem biological phyla [510]. Their earliest role would appear to be the biosynthesis and deactivation of the steroid hormones and the eicosanoids, and their regulatory interplay with receptor activity regulating DNA transcription and replication [ 181. Interference in these vital biological controls by environmental chemicals with spatial conformations similar to the steroid hormones and other endogenous regulators, was minimal so long as the living systems were confined to aquatic systems and the concentrations of environmental lipophilic xenobiotics were low. However, with the advent of the massive increase in atmospheric oxygen, and the associated worldwide conflagration and formation of PAH and other combustion products, a new role fell to the cytochromes P-450, namely, the detoxication of xenobiotics-environmental chemicals which interfered with biological mechanisms. With the evolution of amphibia and terrestrial species, some 300-900 million years ago, this dual function of the cytochromes became grossly inefficient,particularly in that PAH molecules could have been accepted as steroid precursors and metabolically oxygenated to pseudosteroid hormones, thereby disrupting the natural regulation of the DNA. The result was the progressive evolution of major superfamilies of cytochromes to deal specifically with steroid hormone biosynthesis and DNA regulation, on the one hand, and with xenobiotic detoxication on the other. The cytochromes P-448(P450 I) would seem to be a vestigial form of the original primitive bifunctional enzyme, capable of inserting oxygen into conformationally hindered positions for steroid biosynthesis, or into unhindered positions for xenobiotic detoxication; and, as such, they are capable of forming reactive intermediatesthat result in toxicity and malignancy. It is now appreciated that many potentially toxic chemicals manifest their toxicity only after exposure to enzyme system(s) that readily convert them into reactive intermediate(s). This activation process occurs as an alternative to the more frequent process of detoxication; both metabolic processes involve oxygenation of the chemical molecules, and both are catalyzed by the same type of enzymes, namely the mixed-function oxidases, predominately the cytochromes P-450.The cellular population of the different cytochrome P-450 isoenzymes at the time of exposure will thus largely determine the metabolic fate of the chemical, and whether detoxication or toxicity/carcinogenicity occurs. If the chemical itself can also act as an inducer of the cytochrome P-450 system, repeated administration may give rise to effects different from those


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seen following a single administration. The availability of specific, sensitive methods for quantification of the individual cytochrome P-450 proteins in vivo, together with a knowledge of the chemicals they metabolize, will enable the prediction of individual responses to particular drugs and other xenobiotics, and will facilitate the identification of individuals and populations particularly susceptible to the toxic and carcinogenic effects of specific groups of chemicals. This approach, with specific substrate probes for particular cytochrome P-450 isoenzymes, will allow the identification of chemicals in our diet and environment which, acting as inducers or inhibitors of cytochrome P-450 activities, modulate individual capacities to detoxicate xenobiotic chemicals, and hence individual potentials for toxicity and associated disease states, such as malignancy, cardiovascular disease, diabetes, and immunological disease. Such an approach has been employed using the stereoselective and regioselective metabolism of warfarin in phenotyping human hepatic cytochromes P-450; warfarin is a particularly useful substrate probe as it allow quantitative determination of many cytochrome P-450 isoenzymes using only this one single substrate, thus overcoming any differences in pharmacokinetic characteristics which could result from the use of multiple substrates [134]. The ethical difficulties of obtaining human liver biopsies, or other biological material, excludes the relatively easier phenotyping of humans by means of in virro testing with a battery of specific substrates. However, the recently described use of human hair roots merits further investigation [511], and human leucocytes [5 121 with their well-developed endoplasmic reticulum deserve further investigation. Both exhibit EROD activity induced by exposure to polycyclic aromatic hydrocarbons. Apart from the application of this fundamental knowledge concerning substrate specificities of the cytochromes P-450 in understanding the etiology of malignancy and other degenerative disease, and in the identification of susceptible individuals and hazardous chemicals and environments, the ultimate goal would be to design selective inhibitors of the cytochromes P-448 and other activating isoenzymes that would modulate the overall activity of the cytochromes P-450, augmenting detoxication, and inhibiting chemical activation and the deregulation of DNA.

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Microsomal lipid peroxidation: the role of NADPH--cytochrome P450 reductase and cytochrome P450.

Location of functional centers in the microsomal cytochrome P450 system.

Multiple forms of cytochrome P450 in the microsomal monooxygenase system.

Microsomal cytochrome P450 in human brain regions.

Involvement of microsomal NADPH-cytochrome P450 reductase in metabolic reduction of drug ketones.

The cytochrome P450 gene superfamily.

Comparative study of monomeric reconstituted and membrane microsomal monooxygenase systems of the rabbit liver. I. Properties of NADPH-cytochrome P450 reductase and cytochrome P450 LM2 (2B4) monomers.

Liver microsomal lipid enhances the activity and redox coupling of colocalized cytochrome P450 reductase-cytochrome P450 3A4 in nanodiscs.

Characterization and expression of the cytochrome P450 gene family in diamondback moth, Plutella xylostella (L.).

The cytochrome P450 family in the parasitic nematode Haemonchus contortus.

Role of the liver-enriched transcription factor DBP in expression of the cytochrome P450 CYP2C6 gene.

Metabolic activation of the antibacterial agent triclocarban by cytochrome P450 1A1 yielding glutathione adducts.

Effects of avermectin on microsomal cytochrome P450 enzymes in the liver and kidneys of pigeons.

Metabolic activation of the new tricyclic antidepressant tianeptine by human liver cytochrome P450.

Role of ethanol-inducible cytochrome P450 (P450IIE1) in catalysing the free radical activation of aliphatic alcohols.

Polybrominated biphenyls: inducers of hepatic microsomal enzymes and type A cytochrome P450 in the rat.

Role of cytochrome P450 and UDP-glucuronosyltransferases in metabolic pathway of homoegonol in human liver microsomes.

Cytochrome P450 1 family and cancers.

Size- and time-dependent alteration in metabolic activities of human hepatic cytochrome P450 isozymes by gold nanoparticles via microsomal coincubations.

The role of cytochrome P450 epoxygenases in retinal angiogenesis.

Tetrahydrofurane - an inhibitor for ethanol-induced liver microsomal cytochrome P450.

Probing the transmembrane structure and dynamics of microsomal NADPH-cytochrome P450 oxidoreductase by solid-state NMR.

Low temperature studies of microsomal cytochrome P450. determination of dissociation constant of the [Fe2+-CO] form.

Interaction of metyrapone with adrenal microsomal cytochrome P450 in the guinea pig.