Monoayte aryl hydrocarbon hydroxyllase (AWN) ~ctivitymimics Rupffer cell and hepatocyte ARH activity irn an animal model of liver diselsse T. C. PETERSON[

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D~partrnent.~ of'Medick~~e atid Phar-wtacobogy, Cbiaicab Reserarch Centre, Rm. C103, Dallzornsa'e Uaaiversikg., H a l i j x , No%., Cbxpaaeh B3H 4H7

AND

Received January 15, 1991

PETERSON, T. C., WHLIAMS, C. N., and MALRI'J~~LEAN, D. A. 1991. Monocytc aryl hydrocarbon kydroxyhase (AHH) activity mimics Kupffer cell and hepatocyte ABH activity in an animal model of liver disease. Can. 9. Physiol. Pharmacol. 69: 1787 - E 883. We have previeausly reported that rnonocyte aryl hydrocarbon hydrowylase (AHH) activity is depressed in patients with Eiver disease and is decreased more in cirrhosis than in early stagc Eiver disease. To determine if msvaocyte AKH activity reflects liver AHH activity, we studied an animal model of cirrhosis, i.e., yellow phosphoms induced cirrhosis in thc pig. AHH activity was detectable in monocytes isotatsd from peripheral blood of normal pigs (8.32 & 0.13 nanctl mg P . h-', tr = 1I) and was corasparabHe to the level of AHH activity in hepatic K~apffercells istrlated from wedge or needle biopsies of livcrs s f normal pigs (0.38 f 0.21, n = 7). The AHH Bevel in pig Kupffer cells was approximately 10% of the AHH level in baepatocytes and rnicrssornes. To induce liver disease, pigs were administered yellow phosphsems (0.6 mgikg) 5 days per week for 16 weeks. At 4 weeks of treatment, monocyte AHH activity was not different from control and liver histology was normal. Depression of monocyte AHN activity was evident at 8 weeks of treatment when liver fibrosis was seen histologically. At 12 weeks of treatment when histology revealed extensive liver fibrosis and collagen levels were elevated, the level s f rnonocyte AMH activity was decreased 67% compared with controls. Similar changes were observed at 12 weeks in Kupffer cell ARH activity (84% decrease) and hepatocyte AHH activity (70% decrease) con~paredwith controls. Thesc results suggest that monocyte AHH activity reflects liver AMH activity and may be a good indicator of ehangc in liver enzyme hnctit~nin Ikver disease in the pig n~sdels f cirrhosis. Key rvords: aryl hydrocarbon hydroxylase, animal model of liver disease, mc~nmytc,hepatocyke, Kupffer cells.

-'

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PETERSON, T. C.. WILLIAMS, C. N., et MALATJAEEAN, D. A. 1991. Monocyte aryl hydrocarbon hydroxylase (AHH)activity mimics Kupffer cell and hepatocyte AHH activity in an animal model of liver disease. Can. J. Physiol. Pharrmacoi. 69 : 1794-1803. Nous avons rapport6 antdrieurement que lqactivit&aryl-hydrocarbarre hydroxylase (AHH) des moncbcytes est rCduite chez les patients ayant une maladie de h i e et qu'elle est diminuke davantage lors d'une cirrhosc que durant les premi&res phases de 13 maladie de focaie. Pour determiner si l'activitk AHB des monocytes reflkte celk du h i e , nous avons examine un modkle animal de clrrhsse, c-.&-d. sirrhose indarite par le phosphore hlanc chez le porc. L5activi%6 AHH a 6tC dCtectabTe dam Ies rnonoeytes lsolCs Qu sang pCriph6rique de porcs nesrmaux (0,32 f @,I3nmol . m g - 9 h W i ,n = I I ) et a 6t6 conzpal-able au taux d9activit6AHH dans les cellules Krrpffer isolCcs de biopsies de h i e s de POHCS normaux (0,38 f 8,21, n = 7 ) . Le taux d5AHHdans les cellules Ka~pfferde port s'cst term approximativcrnent 2 20% du taux d9AHHdans les hCpatocytes ct les rnicrosomes. Pour induire 1: maladie Emtpatique, du phosptac~rebHanc (O,6 mgikg) a 6tC inject6 aux porss 5 jcxurs par semaine pendant 16 scrnaines. A la eb" semaine de traiternent, E7actiwit6AHB des rnonocytcs n'a pas differ6 de H9activiaC tCrnoin et 15histologiedu foic etait normale. La diminution de 19actiwit6AH%% des yonocytes a 6t6 rnanifeste h la 89em-apaine de traitcment lorsqu'une hisiologie a permis d'observer une fibrose hdpatique. A la 12" semaine de traitement, lorsqkae 19histologiea rCvkl6 une fibrose hkpatique &endue et que les taux de collag5ne avaient augmentt, le taux d'activitk AHH des monocytes a 6tC rCduit de 617% comparative~nentB celui des tkmcsins. Bes variations similaires ont 6% observCes h la 12" semaine de traitenmerat dam 19activie6AHH Qes cellbales de Kupffer (diminution de 86%)et dam 19activit6AHH des hdpatocyres (diminution de 90%) comparativement aux tCmoins. Ces rksultats suggkrent que I'activitC AHH des monocytes reflkte l'activitk AHH daa foie et qu9elle psuerait &re ura bon indicateur de variation de la fanction enzymatique hepatique de ka maladie de foie dans le rnodkle de sirrhose chez le port. Mots c l h : hydrocarbure d5aryl hydroxyiiase, modkle animal d'urme maladie de h i e , monocyte, hepatocyte, ceEI~lesde Kupffer . [Traduit par la r6dactionI

Zntrodnction enzymes are chiefly responsible for this metabshism. One of these enzymes7 aryl hydrocarbon hydroxylase (AHH), has Hepatic metabolism of drugs, chemicals, xenobiL3tjcs8s well as endogenous compounds such steroids ( ~ ~ et ~alad i been ~ repaned to be more greatly depressed than the other P-450 enzymes in a rat model of h i 2 r disease (Murray et ai. 1981) is decreased in liver disease. The cytochrome p-450 1986).

'Author for correspondence. Pnnted In Canada i Irnpr~rnC~ l uC.~nada

AHW activity can be detected in wonocytes isolated from peripheral blood (Peterson 1987; Bast et al. 1974; Pbashne

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et al. 1974) and is depressed in monocytes s f patients with liver disease compared with normal volunteers (Peterson and Wiliarns k987). Recent work from our lab indicates that monocyte AHH activity changes with the progression or resolution of human liver disease (Peterson and Williams 198'7), correlates with serum procc~llagentype 3 peptide (Peterson 1988), and is significantly lower in patients with liver disease who do not survive (Petersan I99 1). AHH may be a sensitive indicztsr s f changes in liver function during disease. The present study was undertaken to determine if rnousocyte AHH activity changes during induction of liver disease in an animal model. The pig provides a good model to study the modulation s f cytochrsme P-458 in liver disease because considerable characterization has been done in this animal (Peterson et al. 1987; Ftajimori et 21. 1986; Williams et al. 1986). Mcsnocyte ABR activity was compared with AHW activity in liver macrophages (i.e., Kupffea cels), I~egatocyteAHH activity as well as the total liver rnicsosomaH AHK activity, to determine if the monocyte AHM activity mimics changes in liver AH%% activity and reflects changes in liver functional status during disease development in this animal model.

&laterials and methods

Negatcscjr~eand Kupfler cell isolation Repatwytes and Kupffer cells were isolated from wedge or needle biopsies of pig liver (2 g) by collagenase perfusion, gravity settling. differential centrifugation, and immune adherence as previously described (Peterson 1989). Microsome prepamtlsra Microsornes were prepared from liver using the ntethod of EIDefraway EI Masry et al. (1944). Ce8lular atrd microsomaE erazyme kactivity: AHH activity ire cells t ~ n d micr~soine~ Macrophage, Kupffer cell, hepatocyte, and rnicrosomal ANH activity was determined in hsmogenates as described previously (Peterson 1984) by an adaptation of the method of Cantre11 and Bresnick (1942) and expressed per milligram of cellular asr microsoma1 protein (Bradford 1976). Macrophages from pig and human blood had similar AHH activity of 0.32 nrnol 3-hydroxybennzopyrene - mg-' cellular protein = h ' (nmol 3-BHBP mg P k-'1 (Peterson 198%).Enzyme assays were done on samples containing 2.5 X lo5 macrophages per reaction. The levels of constitutive AHB activity reported here arc consistent with those reported by others (Cantrell and Bresnick 1972) and are lower than the AHH levels reported for induced Kupffer sells (Cantrek1 and Bresnick 1972) and induced monocytes in culture (Bast et al. 19'74).

AnimaD earad rreamzent schedules Female Duros -Hampshire cross pigs were obtained Rom a local breeder as weanlings (age 5 weeks), allowed to accli~natizefor I week in the Animal Care Facility, and fed Purimla pig chow daily and water ad libitum. The body temperature was maintained during this acclimation period by use of heat lamps. At age 6 weeks the pigs were started on oral yellow phosphorus (0.6 mglkg) once a day for 5 dayslweek. The yellow phosphorus was BjlssoBved in mineral oil to a concentration of 8.1 mg1mL and fed to the animals via a syringe. Control animals received mineral oil alone. Pigs tolerated this route of administgation well with no apparent side effects. Pigs were weighed twice a week and weights recorded. At designated time intervals wedge biopsies were obtained from the pigs for histological cxaminatictn and for isolation of hepatocytes, Kiapffer cells, and preparation s f hepatic microscsrnes. At these times blood sampIes were also obtained by cranial vena caval venipuncture for isolatic~n of monocytes for monosyte AHH activity and also to obtain serum for analysis of standard liver function tests.

Tissues Liver wedge biopsies were taken under general anaesthesia for histological examination. Sections of the liver were fixed in 10% buffered Formalin for histology and stained with Masson9s trichrome. Pigs treated with yellow phosphonls were compared with controls treated with mineral oil. Liver sections taken from pigs treated with yellow phosphorus were divided histcdogically into three stages: (9) early fibrosis, (2) extensive fibrosis, and (3) established cirrhosis as determined by assessing sections seained ~ 4 t hMasson's trichrome for the abnormal presence c~fcollagen indicative of these stages. The appearance of thickened fibrous septia that separate hepatic lobules is characteristic of early fibrosis (stage 1); no bridging was seen in early fibrosis. The widening s f fibrous bands, the presence of irregular septa, the presence of sinarscpidal fibrosis (including periceneral, priseptal and periportal), and con-lplete bridging fibrosis from central no sepal regions and from central tcs portal regions are characteristic of extensive fibrosis (stage 2). Complete bridging fibrosis and nodanlation are characteristic of established cirrhosis (stage 3).

M~.wzocyteisob6~fiormfrom blood Peripheral blood (20 mL) was obtaincd fom each pig and monocyecs were isolated using a modificatism of the method of Boyum (1368). Blood was nixed with RPME-1640 medium and layered over Histopaque (Sigma Chemical Co., St. Louis, MQ) to isolate leankocytes and then washed with WPMI-1640 at 2W x g , for HO min. Finally the pellet was resuspended in Leibovitz (L-15) medium supplemented with 10% fetal calf serum and 10% antibioticlantimycotic solution, and plated and incubated at 37'C for 4 h. After 4 h the medium was replaced with fresh supplemented L-15 medium. At this stage small aliquots (50 pL) of ceIIs were placed on glass slides and fixed in citratc:acetone:methanol for later cytschemical identification of macrophages by the standard method for nonspecific esterase staining (Ksaplow 1981). The remaining isolated cells were incubated for 24 h at 37°C. The monscytes were purified by adherence (Pennline 1981). Cells were incubated in L-15 medium supplemented with I 0 74 fetal calf serum and I0 antibioticlantimycotic solraticsn for 24 h prior to harvest: at this time the cells were more macrophagelike in appearance. Cell monolayers were washed with 1.15% KC1 prior to harvest, and were harvested by scraping with a rubber policeman. Owing to the high stringency of washes, the resulting cell population contained greater than 95% monocytes and was greater than 99% viable. as determined by trypan blue exclusion. Cell yield was determined. Cells were homogenized prior to use in macrophage AHH enzyme assays.

Collagen estimation in livers A recent study by Gascon-Barre et al. (1989) investigated the relationship between morphometric evaluation and coloriometric determination s f hepatic collagen content. Their results revealed a significant correlation ( r = 0.9458; p < 0.801) between morphometric and colorimetric methods of collagen evaluation of liver specimens; both methods significantly distinguished data obtained fronn controls and from cirrhotic rats ( p < 0.0005). According to these authors the severity of hepatic collagen infiltration can be determined on a single liver specimen if that section is taken from a major lobe of tl-ee liver and if sufficient numbers of animals ( n = 10) are studied to avoid occasional sampling errors. A liver specimen was obtained from the core of the median lobe, fixed in 18% buffered Formalin, and embedded in paraffin. Two sections of liver adjacent t s those used for histology were cut at approximately 15 pm thick, resulting in a tissue area of 50- 100 mm'. Evaluation of the collagen content was based cdra the method published by Lopez-de Leon and Rojkind (1985). The liver sections were deparaffinized with successive incubation with toluene, toluene~ethanol (1:5), 50% aqueous ethanol, and water. The samples were then kept in distilled water at 4OC and stained as follows. Individual slices were incubated in aluminum foil-covered test tubes in the presence of a 0.84% solution of Fast green FCF in saturated picric for 15 min. The sections were then washed thoroughly with distilled water until the supernatant was clear; they were then incubated in the presencc

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PETERSON ET AL.

s f Fast green FCF 0.1 % and Sirius red F3B 0.04% for 30 min and washed as described. above. One rnillilitre of 0.05 M NaOH in 50% aqueous methanol was then added. and each tube was gently mixed. The eluted color was read in a spectrophotonseter at 530 and 624 nm (corresponding to the maximal absorbance of Sirius red and Fast green, respectively). To quantitate the abssrbanacc found for each dye the color equivalences (Lopez-de Leon and Wqjkind 1985), i.e., 2.08 for Fast green and 38.4 f i r Sirius red, were Lsed. Noncollagenous protein determination was obtained as noncoklagenaus protein (mg) = absorbance 60512.08. Collagenous protein was determined as callagen (pg) = absorbance 540 nrn - 0.26 absorbance 605 nm138.4. The collagen content was calculated. as collagen content (pglpg total protein) = pg collagenlyg collagen f pg noncollagenous protein. Using these experimental conditions, maximal, seable, and reproducible absorbance was obtained for both dyes (Gascon-BarrC et al. 1989).

Sta~adardliver J%BZC'~~OB;P FeSdS Standard serum biochelraical liver function tests were done on semm saampkes obtained from pigs at the time intervals indicated (0, 4, 8, 12, 16 weeks), and results froln pigs treated with yellow pkosphorus were compared with controls treated with mineral oil for the same time period. Scmm liver function tests included albumin, total protein, aianine aminotransferase activity, aspartate aminstransferase activity, y-gluramyltranspepeidase activity, and total and direct bilirubin and alkaline phosphatase activity. These were analysed by sequential multiple analysis.

TABLE1. Ary1 hydrocarbon hydrsxylase (AMH) activity in hepatic microsomes, hepatocytes, K ~ p f k cells, r and rnokeocytes

AHH activity (nmol 3-OMBP - mg-I P . h-I) Enzyme source (" = 4, Hepatocytes (n = 6) Kupffer cells (n = 7 ) Monoc~tes (BZ = HI)

YP

Control

% decrease

3.391.3

2.7k8.7

0.82f 8.3""

78

0.4f 0.2"

0.052 f0.05"*

86

0.3f Q.I*

0.1 f0.047"*

67

Noae: Each value represents the mean r SE of the nurnber of anirniils indicated in paremtkcses. ControUs are treated for the same 12-week time period with mineral oil (vehicle). 3-OHBPI 3 hydraxybenzopyrerae; m g P, rng prorein; YP, yelkow phosphorus. * p < 0.05. statistisalty significant difference compared with Expatic rnicraaomes and hepatocy tes. **p < 0.05, statistically significant difference compared with control.

AHW activity

Sfatistical analysi.~ An unpaired Student's b-test was used to campare two variabkes, and a Student-Newman-Meul's test was used when name than two variables are compared (Zar 1994).

Results Msncssyte, Kupffer cell, hepatsc yte , and microsomal AHH activity were determined in mineral oil treated controls; results are expressed as mean k SE (Table I). In control animals, rnowocyte AHH activity did not differ from Kupffer cell AHH activity but was significantly Iower than hepatocyte AHH activity. Kupffer cell AHH activity was approximately 18% s f hepatocyte AHH activity. Similar results have been reported for the relative AHH activity in Kupffer cells and hepatocyks in rats (Cantrell and Bresralsk 1972) and mice (Peterson and Renton 1984). Hepatscyte AHH activity did wat differ f r o n ~micras~malAHH activity and all subsequent assays were restricted to cellular AWH activity. AHH activity was significantly decreased in all c e l types: hepatocytes, Kaapffer sells, and monocytes in pigs treated with yellsw phosphorus for 12 weeks compared witk the corresponding 12-week controls (Table 1). Similar results were obtained in repeated experiments. No change in monocyte AHW activity was evident after 4 weeks of treatment. After 8 weeks of treatment witk yellow phosphorus there was a significant decrease in ra~cbnocyteAHH activity compared with control pretreatment activity. The AHH activity remained significantly decreased after 12 and 16 weeks of yellsw phosphorus (Fig. 1). The decrease in Kupffer cell AHH activity paralleled a decrease in monocyte AHM activity. Ktapffer cell AHH activity was significantly decreased 74% at 8 weeks (0.1 + 0.035 nmol mg-I protein * h-') and remained significantly decreased 86 7%at 1%weeks and 99 % at 16 weeks after treatment compared with control pretreatment activity (8.4 f 8.2 nmol * mgg8protein h-K). Hepatscyte AHH activity was significantly decreased '30% at 12 weeks (0.82 & 0.3) and remained significantly decreased ('38%)at 16 weeks after treatment with yellow phosphorus compared with control pretreatment activity (2.7 f 9.7).

Contr01

4 weeks YP 8 weeBtsYP

12 wseks VP 16 weeks YP

FIG. 1. Change in monocyte AKH activity over 16 weeks of yelle~w phsspkoms (YP) treatment. Each bar represents the mean f SE of six pigs. * p < 8.05, statistically significant difference compared with ssntrsls; each pig served as its own ccdntrol, i.e., control was pretreatment activity.

Standard liver function tests were done initially and repeated at 4, 8, 12, and 16 weeks of yellow phosphcsrus treatment. Preliminary studies indicated that mineral oil alone did not affect standard liver function tests compared with untreated animals. At 4 weeks all liver function tests were normal and did not differ from pretreatment levels except for alkaline phssphatase, which showed a gradual increase over the first 8 weeks. Since this pattern was also seen in control animals, the increase in alkaline phosphahse is likely related to the growth of the gig. Food eorasumpticsn and weight gain in the animals receiving yellsw phosphorus did not differ from controls throughout the experiment (Peterson et al. 1987). Between 8 and 12 weeks, moderate changes in liver function tests were observed in some of the animals, but most of these changes were not significant compared witk controls; only serum albumin levels at 12 weeks were found to be significantly decreased in yellow phosphorus treated animals compared with controls. All liver h n c d s n test results are reported in Table 2. Changes occurred in the following tests: serum albumin concentration was significantly decreased (p < 8.05) from 36.4 f E . 3 to 30.1 _+ 0.4 g/L in gigs treated for 16 weeks with

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TABLE 2 . Serum biochemical liver fianactionm test results for pigs treated with yellow phosphorus (YP) or vehicle (mineral oil) for $, 12, and 16 weeks

12 weeks

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8 wreeks

Serum albumin (g/E) Total protein (g/L) Total bilisktbiw (pM/E) GGT (U/L) AST (U/E) ALT (U/E) AP &U/L)

16 weeks

Mineral oil

YP

Mineral oil

YP

Mineral nil

YP

35.511.7

31.3k1.2

35k0.7

31.5f 0.6"

36.4kI - 3

38f 0.4"

51-i-1

48t2

50k0.3

51 &2

53k3

54k1.4

1.7 f0.3 36p3

2813 65+ 14

276k 12

13+0.3 43i-2 24 & 4 54f 22 259k-26

li-0 37k5 51i16 6Bp9 230k 16

2k0.6

53p 80 46C7 79f 7 288f43

1.3k0.3

2.70.9

34f 2 33 k3 5Qk5

92 IB* 63k11" 54 & 4 228f14

214kI1

+

Nore: Yellow phosphcrrlas (0.6 mglkg) was administered orally 5 dayslweck for the number of weeks shown: control animals received the same volume of vehicle (mineral oil). CGT, y-gluhmyBtraraspeptidase: ALT, alaninc: an?inotransfer;asc; AP. aCkaline phosphatase: .As%,aspartale aminotrarasfemse. *p < 0.05. statistically significant diffcreace from corresponding coneso!.

yellow phssphsrus compared with mineral oil treated controls; serum protein and bilirubin concentratisns in yellow phosphorus treated pigs were not different from control: y-ghtarnyltranspeptidase activity was significantly elevated ( p < 0.05) from 34 -1- 2 to 92 _g 49 UIE; and aspartate aminotrannsferase activity was significantly elevated ( p < 0.05) from 33 f 3 to 63 p 11 U/L in animals treated for I & weeks with yellow phosphorus compared with animals treated for 16 weeks with mineral oil. Alanine aaninotransferase activity in yellow phssphoms treated pigs did not differ from controll. Histological elevation s f liver sections stained with Masson's trichrsme taken from liver biopsies after 8 weeks of yellow phosphorus treatment revealed early fibrosis characterized by the zppearance s f thickened fibrous septa (not shown) as described in the Methods section. After 12 weeks s f yellow phosphoms treatment the histological evaluation of liver sections shined with Masson's trichrome showed extensive fibrosis characterized by incomplete bridging fibrosis, widened Ebrous bands, thickened septa, irregular septa, and sinusai&a1 fibrosis including periseptd, pericentrall , and periportal (Fig. 2A). HistolsgicaH examination s f liver sections taken from control pigs after 12 weeks of mineral oil treatment revealed normal pig liver architecture with delicate fibrous septa separating normal hepatic lobules (Fig. 2B). Preliminary studies had also indicated that mineral oil alone did not affect liver histology compared with untreated animals (not shown). Mineral oil treated pigs were included in all experiments. Liver sections taken from pigs treated with yellsw phssphsrus for 16 weeks have established cirrhosis characterized by noduZatisn and complete bridging fibrosis including central to septal bridging and central to portal bridging (not shown). Pigs treated with mineral oil for 16 weeks had histologically normal livers. Liver sections obtained from a group of animals treated with yellow phosphorus or mineral oili for I2 weeks were used for collagen determination. Collagen estimation by colsrimetrie methods indicated that the yellow phosphorus treated animals had signiEcantly higher levels of collagenous protein: 8,032 f 0.8035 pg colllagenlyg noncollagenous protein in their liver sectisns compared with animals treated with mineral sili 0.018 -t- 8.8008 ( p 0.019). The results indicated that the yellow phosphorus treated animals showed an 88% increase in

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collagen content in the liver compared with the mineral sii treated controls . The gradual, progressive development of liver disease in this animal model is reflected in histology and serum liver function tests (LFTs), and results reported here indicate that this is also reflected in a noninvasive peripheral blood test. In the animal model of liver disease based in this study, female Dursc - Hampshire cross pigs showed no histological changes after 4 weeks of treatment, early fibrosis by 8 weeks of treatment. extensive fibrosis by % 2 weeks, and established cirrhosis as defined by nodulakion by 16 weeks of treatment with yellow phosphorus (Peterson et a]. 1987). No changes in serum LPTs were observed after 4 weeks of yellow phosphorus treatment compared with contro%s,while after 16 weeks s f yellow phosphorus treatment several serum LHTs were abnsrma1 in all pigs and were significantly different from controls. After 4 weeks of yelllow phosphoms treatment monocyte AHH activity did not differ from controls, but after 8 weeks of yeilow phosghoms treatment both the monocyte and Kupffer eel% AHH activities were significantly decreased compared with controls. Changes in monocyte ARH activity were evident prior to observed changes in serum LFTs. This supports an earlier report in which two patients with normal serum LFTs had decreased monscyte AHH activity suggestive of liver disease. Their biopsies revealed extensive fibrosis, suggesting that monocyte AHH activity in these patients was a better indicator s f liver disease than EFTS (Peterson and Wiliams 1987). Unlike other toxins such as carbon tetrachloride (Trey et al. 1969; Lamson et 21. 1924; Leviw et al. E973), yellow phosphoms did not appear t s directly affect cytochron~eP-450 mediated drug metabolism, i.e., P-450 mediated enzyme activities were normal after 4 weeks of yellow phosphoms treatment. Cytmhsome P-450 enzyme activity was depressed after 8 weeks of yellsw phosphorus treatment at a time when the liver histoleggy revealed fibrotic changes. These results suggest that the decrease in AHH may relate to liver fi~nctionalchange and is not likely due t s a direct effect s f yellow phosphorus on AHH enzyme activity. Our resulks also suggest that the effect on ranonscyte ABH activity is unlikely t s be due to a direct

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PETERSON ET AL..

FIG.2. (A) Liver section taken from a pig treated for 12 weeks with yeElow phosphorus. After 12 weeks of ~ C I I O Wpl-aosphorus treatanent the kistologicd evaluation of liver section stained with kr~%~assc?n's trichrume showed extensive fibrosis characterized by incomplete bridging fibrosis (snaal arrowheads). widened fibrous bands of thickened, irregular septa (large arrowheads), and sinusoidal fibrosis including perisepeal. perisentrd, and periportal. (B) Histological examination of a liver section taken from control pig after 12 weeks of mineral oil treatment revealed normal gig liver architecture with delicate fibrous septi (large arrowheads) separating laorlnal hepatic lobules.

toxic effect of yellow phosphorus on rnowwytes because the monocytes tested for AHH activity did not show decreased AHH activity until 8 weeks after treatment and also showed similar viability to controls. Results presented here further indicate that mowocyte, Mupffer cell, and hepatoeyte AHH activity were significantly decreased at 12 weeks whew all pig livers showed marked fibrosis and the AHH activity remained significantly decreased after E 6 weeks of yellow phosphoms treatment when Ristnlsgy revealed established cirrhosis in all pig livers. These results suggest that decreased monocyte AHH activity reflects decreased Kupffer cell and hepatocyte AHH activity during progression of lives disease. Kupffer cell AHH activity is totally depressed after 16 weeks sf treatment. The mechanism for depression of rnsnocyte, Kupffer cell, and hepatocyte AHH activity in liver disease is presently unknown but is under investigation. Estimation of collagen using a colorimetric method recently reported by Gascsn-Barn5 and colleagues (1989) revealed that collagen per se was significantly elevated in liver sections obtained from animals treated with yellow phosphorus for 12 weeks. Reports by Gascow-Barre and colleagues indicated that the cslsrimetric method was as good as the morphometric

method and provided an adequate estimation of collagen content in liver sections. This marked elevation in collagenssus protein content in liver sections strongly supports the histslsgieal changes observed with Masson's trichrome staining. The animal model of liver disease we have developed Is a modification of the original model of Ma6lory (19331, who reported that yellow pheesphsms (8-6-k mg/kg orally) produced cirrhosis in rabbits and guinea pigs, and that lower doses would produce the degenerative changes characteristic of alcoholic cirrhosis. Long-term low-dose yellow phosphorus treatment in pigs reproducibly produces cirrhosis in 16 weeks, Yellow phosphorus ingestion in humans results in liver eirrhosis (Marin et al. 197 H ; Fletcher et aI. 1963)- Swine have been US& extensively as animal models of alcoholism (Tumbleson and Dexter I986BS liver mitockondrlal response to ethanol (Sun et al. I986), liver transplant (White and Lunney 1979), fetal alcohol syndrome (Tumbkeson et al. 19861, collagen bissynthesis (Hek&s et ai. k986), drug meeabo8ism (Fujirnori et al. B986), and bile acid synthesis (Bupont et al. 1986). The hepatic histology of the pig is well characterized (Banks 1981; Elias and Scherrick 1969; Trautmann and Fdebiger 195%)and the dmg-metabolizing enzyme activities are well defined (Fujirnori et ale 1986; Peggins et al. 1886; FreudenthaH et al.

1802

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1975; Short and Davis 1970; Kearns et al. 1986; Dvorchik

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et al. 198%).This model has a reasonable time to induction of

fibrosis and cirrhosis, allows for repeated wedge biopsies over the course of induction of the disease, and can be divided histoIsgicalEy into three stages: ( 1 ) early fibrosis, (2) extensive fibrosis, and (3) established cirrhosis. This animal model provides a relatively large blood volume for isolation s f monocytes (Peterson 1987) or for pharmacokinetic drug studies. In summary, we report that monocyte AHB activity is significantly depressed in a highly reproducible animal madel of lives disease and appears to parallel decreases in this enzyme in the liver when hiistology and collagen estimation indicate presence 0%f'ibrosis and may precede changes in standard liver function tests.

This work was supported by the Dalhsusie Medical Research Foundation and a research grant from the Medical Research Council of Canada. BAKKS,W. 5. 1981. Hepatic sim~usoids.Bn Applied veterinary histology. William & Wilkins, Baltimore, MD. pp. 412 -414. BAST,W. C., OKUDA, T., PLOTKIN, E., TARONE, W., WAPP,H. %.,and G ~ t a o l M. ~ ,Vo 1976. Des9e%opanzentof an assay for arylkydrocrrrbsn hydroraylase in human peripheral blood morqocytes. Cancer Wes. 36: 1963- 1974. BOYUM,A. 1968. Isolation of mononuclear cells and granulocytes froan human blood. Scatad. 1. CHin. Lab. Invest. 97: 37. BRADFORD, M.Ma1976. A rapid and sensitive method for the qua~titaf on of microgram quantities of protein utilizing the principle of protein-dye binding. a d . Biochern. 72: 248 - 254. BWODEE, M . J., BOOBIS.A. R., BUEPITT,C. J.. et al. 1981. Influence of liver disease and environmental factors on hepatic rnonsoxygenase activity i ~vitrs. , Eur. S. Clin. Pharmaso1. 20: 39 -46. CANT RELL, E., and B~ss~.arcag, E. 1972. Benzopyreaae hydroxy lase activity in isolated parenchymal and nonparenchymal cells of rat liver. 9. Cell. Biol. 52: 316-321. DUPOPET, J., OH, S. Y . , O'DBEN,E., et al. 1986. Synthesis and disappearance CPE cholcstesol and bile acids in miniature swine. PFI Swine in bionnedical research. Vol. 2. Ellired hy M. E. Tunibleson. Plenum Press, New York. pp. $21 -838. DVORCHIK, B. H. 1981. Non-human primates as animal models for the study of fetal hepatic drug metabolism. IFLDrug metabolism in the in-srnrrture human. Edited by L.F. Soyka and G.P. Redmoa~eb. Raven Press, New York pp. 145 - 162. ELDEFRAWRY EL MASRY,S., COBEN,S., and ~ ~ N N E R I G. N GJ. , 1974. Sex-dependent differences in dtng anetabolisn-a in the rat. Drug Meaab. Dispass. 2: 267 -278. ELIAS,H., and SCHERRICK, J. @. I969*Mopholngy of the liver. Academic Press, New York. pp. 125- 136. FLEFCHER, G. E , and GALAMBOS, J. T. 1963. PA6~sphoruspoisoning in humans. Arch. Intern. Med. 112: 846-852. FREUDENTHAL, R., LEBER,P.,EMMEWLING. D., et aE. 1975. Characterization of the hepatic microsomal mixed fu~hsfcti(~n oxidase enzyme system in miniature pigs. Drug Metab. Dispos. 4: 25 -27. FBIJIMORI, K., TAKABASHI, A . , XUMATA, H., and TAKANAKA, A. 1986. Drug metabolizing enzyme system of Gottingen miniature pig. In SK in% in biomedical research. Val. 1. Edited 6; M . E. Tumbleson. Plenum Press, New York. pp. 533 -548. G a s c o ~ - B a ~M., ~ k , hue^, P-M., BELGIORNO, J., PL,OUIRDE, V., and CBULOMBE, I?. A. 1989. Estimation of collagen content in liver specimens. Vdsiation Hmong animals and among hepatic lobes in cirrhotic rats. J . Histochem. Cytochem. 37: 373 - 381. HEBDA,P. A , KAO.W., MERFZ,PoM . , and EAGU~STEEN, W. H.1986. Collagen biosynthesis and collagen content as a measuse of dermal healing in cxperimenral wou~ldsin domestic swine. In Swi~sein

biomedical research. VoB. 1 - Edited by M. E. T ~ r n b ~ e sPlenum ~n. Press, New York. pp. 383-312. KAPLOW?E. S. 1881. Cytochemical identification of mc~nonuslear macrophages. Bn Manual of macrophage methodology. Edibed by H. B. P%erscowitz,H. J. Holden, J. A. Bellant, and A. 61.maffar. Immunology Series. VoE. 13- Marcel Dekker, New York. pp. 199 -207. KEARNS,G. &.. HILL, D. E., %ORNBERRY, c. PB%., and ~ L J N Gj., E 1986. Developmental pharmac6~kinetiCsof theophylline and caffeine in neonatal piglets. In Swine in biomedical research. Vcll. 2. Edited by M. E. Taan~bleson. Plenum Press. New York. pp. E383- 1312. LAMSON, P. D., GARDNER, G. W., GUS'WFSQN, R. M.,et aJ. 1924. The pharmacology and toxicology EPI' carbon tetrachloride. I. Pharmacol. Exp. Thee. 22: 15 -288. LEYIN,We*Lu, A. Ye H., JACOBSON, M.. KUNTZMAN, W., PWER, 9. L., and MCCOY,P. B. 1973. Lipid peroxidation and the degradation of cytoclmrorne P-450 heme. Arch. Biochem. Biophys. 158: 842 - 852. Lopez-DE LEON,A , , and ROJKHND, M. 1985. A simple micron~ethesd for collagen and total protein determination in Formalin-fixed paraffin-einbedded sections. 4. Histoehem. Cytcochem. 33: '537 743. MAL~RY 'F-, B. 1933. Bhorphoms and alcoholic c i r r h ~ s i sAm. ~ J. Pathol. 91 557 -567. MARIN,G. A . , M O N ~ Y C A ., A.,SIERRA, %.L., and SENIOR, J. W. 1931. Evaluation of coflicosteroid and exchange transhsion treatment of acute yellow phosphorus intoxication. N. Engl. $. Med. 284: 125- 128. MURRAY,M . , ZALUZNY, L.,and ??A~RELL, 6.C . 1986. Dmg metabolism in cirrhosis; selective changes in cytochrorne P-450 isozyrnes in the cholilake deficient rat model. Biochem. Bharmacnl. 35: 1817. PEGGINS,9. O., SHIPLEY, L.A . , and WEINBR,M. 1986. Effects of age on hepatic dmg metabolism in miniature swine. 6~Swine in biomedical research. V01. 1. Edbbed by M. E. TumbHeson. Plenum Press, New York. ppo 39 -44. PEWNLINE, K. J. 7981. Adherence to plastic and glass surfaces. In hlanual of macrophage mebKodoEogy. Edl'frd by H. B. Mcrscowitz, H. 9. Molden, J. A. Bellant, and A. Ghafhr. Immunology Series. V01. 13. Marcel Dekker, New Yo&. pp. 161- 169. PETERSON, T. C. 1987. Drug metabolizing enzymes in rat, mouse, pig and human macl-ophages. Biochem. Pharrnacol. 36: 39 11 - 39 16. PETERSON, T.C. 1988. Procollagen-RI-peptide and mowocyte AHH activity in patients with liver disease. MepatoEogy, 8: E 3 1 1. 1991. Msnocyte aryl hydrocarbon Pkydroxylase activity and mortality. Clin. Invest. Med. 84: 32-76. PETERSON, T. C., and WILLIAMS, C . N. 1987~~. Depression of monocyte AHH activity in patients with liver disease: possible invokvement of maerasphage Fdctors. Hcpatolsgy. 7: 333 - 337. 1983b. Changes in nlonalcyte AHH activity with progression and resolution of liver disease. Hegatolasgy, 7: 1166. PETERSON, T, C., and RENTON,K,Wo 1984. Ekpression of cytochrome B-450 dependent drug biotransformation in hepatwytes a f er the activation of the reticuloendothelial system by dextrran sulfaa. 9 . Pharmacol. Exp. Ther. 229: 299. PETERSON, T. C., MAEATJALIAN, B., and WILLBAMS, C. W. 1987. Monocyte AHH activity in a pig model of cirrhosis: does the Bnonscyte enzyme reflect liver enzyme changes'? Clin. %naicst. MeQ. 10: B131. P~ASHNE, M . , B R ~ ' ~ H EE., R SAXLINE, , S. G., and COHEN,S. N. 1974. Aqrl hydrercarbc~nRydrcsxylase induction in mouse peritoneal macroghages and blood-derived human n~acmphages.Bproc. SQC.EXP. Bid. Med. 845: 585-589. SHORT,C, R., and DAWS,&. E. 1970. Perinatal development of dmg metabolizing enzyme activity in swine. J . Pharmacol . Exp . Ther . 174: 185- 196. SUN,A . Y.: TUMBLESON. M. E..and DEXTER,%.D. 1986. Effect of

Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by University of North Dakota on 12/19/14 For personal use only.

M . E., DEXTER. J. B., and LOHMAN, K. 1986. Fetal ethanol on liver mitochondria1 Ca2+ uptake. In Swine in bio~~ledi- TUMBLESON, alcohol syndrome in miniature swine. 8~ Swine in biomedical cal research. Val. 2. Edited by M. E. Turnbleson. Plenum Press, research. V01. I. Edi~edby M. E. Turnblesona. Plenum Press, New New York. pp. 907-914. T ~ ~ T ~ I A NA.. N , and FIEBIGER, J. 1952. The liver. Bn Fundamentals York. pp. 597-609. of the histology sf domestic animals. Cornstock, New York. pp. WHITE, F., and LUJNNEY, J. 1979. Transplantation in pigs. 2 19 - 22'7. Transplant. Proc. 11: 1170-1173. ~ E YC., , GARCIA, F. G., KING,N.N7., et al. 1969. Massive necrosis WILLHAMS, @. NBa., M A I . . ~ A L I A D., N , and PETERSON, T. C. 1986. Yellow phosphorus induced cirn kssis ian the pig. Broc. Falk Symp. in the monkey: the effects of exchange blood transfusions on h l minant liver failure. J. Lab. Clin. MeQ. 93: 784-793. 44: 1984. TWMBLESON, M. E., and DEX-TEW, 9. D. B 986. Plasma ethanol concenZAW,J. M. 1974. Biostatistisal analysis. Prentice-Hall, Englewood trations and volaantary dietary ethanol coeasumptions for 3 years by Cliffs. NS. male and female miniature swine fed diets to effect a marginal protein deficiency. Hn Swine in biomedical research. %l. 1. Edited by M. E. Tumbleson. Plenum Press, New Yortd. pp. 583 -596.

Monocyte aryl hydrocarbon hydroxylase (AHH) activity mimics Kupffer cell and hepatocyte AHH activity in an animal model of liver disease.

We have previously reported that monocyte aryl hydrocarbon hydroxylase (AHH) activity is depressed in patients with liver disease and is decreased mor...
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