DRUG AND CHEMICAL TOXICOLOGY, 1 4 ( 4 ) , 319-342 ( 1 9 9 1 )
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THE EFFECT OF TETRAHYDROFURAN ON BIOLOGICAL SYSTEMS: DOES A HEPATOTOXIC POTENTIAL EXIST ?
David E. Moody Center for Human Toxicology, Department of Pharmacology and Toxicology, University of Utah College of Pharmacy, Salt Lake City, UT 84108
ABSTRACT Tetrahydrofuran (THF) is a widely used solvent in industry and research. THF is a weak toxin, with approximate acute L D ~ o in s the range of 2 to 3 gkg, 8 to 20 mg/L, and 800 mgkg following oral, inhalatlon, and i.v. routes, respectively. The two primary signs of intoxication are narcosis and hepatocellular dysfunction, both occurring at doses of approximately one-half of the lethal dose. Little is known concerning the pharmacokinetics of THF. No evidence exists for genotoxicity due to THF. Ongoing carcinogenicity bioassay tests sponsored by the National Toxicology Program have not yet been completed. Two significant interactions of THF with cellular components have been studied; first, THF inhibits a number of enzymatic reactions at concentrationsranging from 10 to 100 mM. Most notably, THF is an inhibitor of a number of cytochrome P450 (P450) dependent mixed function oxidase activities, with a particular affinity for the alcohol-inducedisozyme (P450IIEl). Second, THF has been noted to enhance the toxic action of a number of compounds (i.e. solvent effect), in particular stimulating the more rapid absorption of reactive metabolites. A third factor to be considered is THF's ability to form peroxides upon standing. Little is known concerning the toxicity of THF peroxides. Therefore, while there is little evidence to suggest that THF would be a direct (Type I) hepatotoxin at relatively low doses, its ability to inhibit drug-metabolizing reactions (perhaps more so in alcohol users), and enhance the absorbance of reactive metabolites, are relatively unexplored avenues for THF to contribute to a hepatotoxic response. As a great deal of the human hepatotoxicity (Type 11) is "idiosyncratic"and cannot be readily reproduced in experimental animals, this potential route of hepatotoxicity should be considered in future evaluations of human exposure to THF. 319 Copyright 0 1991 b y Marcel Dekker, Inc.
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320
I " Tetrahydrofuran (THF), a four carbon cyclic ether, is a colorless flammable Drug and Chemical Toxicology Downloaded from informahealthcare.com by Nyu Medical Center on 11/09/14 For personal use only.
liquid with a smell similar to acetone which is widely employed as a solvent in industrial, pharmaceutical and research settings. The solvent qualities of THF are characterized by its high solubility in water and its ability to complex solutes through hydrogen bonding.' A saturated, unsubstituted furan, THF is a relatively nonreactive ether, with the exception, that peroxides of THF will form upon exposure to oxygen and sunlight. Additional characteristics and general information about THF are summarized in Table 1. The largest use of THF is as an industrial solvent, primarily for dissolution of plastic resins and as a reaction medium. Approximately 150 million lbs. of THF
are produced annually in the U.S. resulting in an estimated exposure of 90,OOO workers.2 The industrial use of THF leads to potential human exposure, through both occupation and generation of industrial wastes. In regard to the latter, THF has been identified in samples from toxic waste sites,3 in drinking water? and in the milk of mothers living in industrial
environment^.^
In addition, THF is
approved as a pharmaceutical solvent, allowing exposure from residues or direct application. Direct application is becoming a greater potential, as THF is employed in novel drug delivery formulations. The purpose of the following report was to review the literature concerning the biological effects of THF. Particular attention was paid to the potential for THF to cause hepatotoxicity.
METHODS Literature citations were collected following Med-line and Tox-line searchs for articles related to the "Biological effects of THF on mammalian systems". These
E F F E C T O F TETRAHYDROFURAN ON B I O L O G I C A L SYSTEMS
321
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TABLE 1. General Information About Tetrahydmfuran (THF)
Synonyms
Furan, tetrah ydro-(Chemical Abstracts); Cyclo-tetramethyleneoxide; butane, 1,4-epoxy ;and butylene oxide
CAS #
109-99-9
RTECS #
Lu5950000
Molecular formula
C4H80
Molecular weight
72.1
Boiling point
65 - 66 O C
Vapour pressure
114 mm Hg at 15 O C
Specific gravity
0.891
Water solubility
30 g at 25 O C
Conversion factors
1 ppm = 2.94 mg/m3 1 mg/L = 340 ppm 1% (v/v) = 124 mM
Tetrahydmfuran WF)
citations were supplemented by abstracts listed in Chem Abstracts (1964 - 1989) under "Furan, tetrahydro- : biological effects". Literature was used if the entire copy of the paper was available at the local libraries, or an abstract was available and contained sufficient data in itself for evaluation.
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There is a dearth of knowledge concerning the p h m a c o - (toxico-) kinetics of
THF. A single study demonstrated that in rats exposed by inhalation, THF accumulated in the brain and fat tissue samples collected (other tissue were not studied), with saturation following 8 (lower doses) to 2 (high dose) weeks of exposure, and subsequent decreases in the body b ~ r d e n .This ~ study, within the realm of the current search, was the only data available on tissue levels of THF (i.e. not even blood levels are available) after any route of administration. Likewise, essentially no published experiments concerning the metabolism of THF exist. Fujita conducted a series of experiments on the tetrahydrofurfuryl mercaptan moiety which demonstrated the saturated furan ring can be cleaved by oxidative metabolism, and Boyd has reviewed a number of studies which consider the metabolism of unsaturated, substituted furans, which may, or may not, apply to the saturated, unsubstituted THF. Kawalek and Anders lo have shown that incubation of THF with liver 9000 x g supernatant from rats pretreated with Aroclor or phenobarbital does result in formaldehyde generation, a common product of mixed function oxidase reactions. The metabolites of THF in biological systems have not yet been elucidated.
The early studies on the biological effects of THF in the 1930s focused on its anesthetic activity, but also included one of the first LD50 determinations for this
E F F E C T O F TETRAHYDROFURAN ON B I O L O G I C A L SYSTEMS
323
TABLE 2. Lethality of THF
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Exposure Ref. Species ~
Strain
Sex
Dose
Route
Duration
inhal.
NA
LC64 LC50 LC16
12.2 mg/L 8.3 mgR. 5.6 m g L
oral
14 d
LD50
2.05 g k g 3.21 glkg 2.85 glkg
oral
single dose
inhal.
2h
LDlOO LDmin LDlOO LDmin LDlOO LDmin
5.0 glkg 3.0 glkg 56-103 m g L 28-55 mgjL 24-29 mg/L 18-21 mg/L
Parameter
~
15.
Rats
Albino
16.
Rats
SD
17.
Rats
NA
NA
M(16-50g) M (80-160 8) M (300-470 g) NA
Mice
NA
NA
inhal.
2h
12.
Mice
NA
NA
inhal.
2h
LD50
72 m a
18.
Mice
NA
NA
i.v.
2h
LD5O
759 mglkg
19.
NA Mice Rats Guinea Pigs
NA
oral
single dose
LD5O LD50 LD50
20.
Mice
NA
NA
LD50
2.50 glkg
Rats
NA
NA
i.p. (20% single dose in olive oil) i.p. (20% in saline) inhal. 3h
MLC
1.90 g k g 2.90 g/kg 61.8 mg/L
LC50
2460 rn@
No effect Min. effect
216 mgR. 367 mgR.
21.
Fish
Fathead minnows
in water
96 h
22.
Fish
Fathead minnows
inwater 33d post-fertilization
(Abbr. used: NA - not available; SD - Sprague-Dawley;M - male; F - female; inhal.- inhalation.)
TABLE 3. Gross and Histological Changes Associated with Exposure to THF ~~
Exposure Ref. Species Strain Sex Route Duration
Dose
Observation
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~~~
15.
Rats
Albino NA
17.
Mice
NA
19.
NA
Rats
NA
NA
Mice
NA
NA
inhal.
NA
5.6 - 12.2 mg/L
-Focal proliferative response in liver (like cirrhosis)
oral
acute
5&3gPcg
- Clinical symptoms within
inhal. inhal. inhal. inhal.
2h 24-29mgL 2h 56- 103mg/L 2h/d;2mt 6 - 8 m g / L 4 h/d;6mt 1 -2mg/L
oral
5mt
5&10mglkg
3-5 min.: somnolence, bradypnea, cyanosis; necrosis of gastric mucosa, enteritis; endemitis cerebri; hemorragic and necrotic foci in liver, kidneys, and spleen. - Similar to oral, but more pronounced lung changes. - Some decrease in body weight and slight histological changes. - Leukocytosis, depression of blood pressure and some proliferative changes in lung.
- No harmful effects at 5 m a g . Growth of mice inhibited at 10 mag.
Rats Rabbits inhal.
constant;3mt 2 u g L
26.
Rats
NA
NA
27.
Rats
SD
M drinking 28d & F water
1.10, loo,& l,OOOmg/L
- Some loss of liver function. - No clinical signs. - No weight loss. - No change in mixed
function oxidase activities. - At high dose, mild histological changes to thyroid, liver, and kidney tubule cells.
24.
Rats
NA
M
inhal. inhal. inhal.
single 44.1 mg/L 30 min/d; 7d 44.1 mg/L 60 min/d; 8 wk 8.8 mg/L
- Irritation, skin and mucous membranes in all groups. - Papillary hyperplasia of lung. - No changes in serum GOT, GPT & AP.
28.
Rats
NA
NA
inhal.
4 h/d; 12 wk 0.29 14.7 mgR.
- Mucousal irritation at all doses. Serum liver function tests altered at 2.9 mgn. Pulmonary mucousal, wbc counts, and liver function altered at 14.7 mg/L. (Con't)
EFFECT OF T E T R A H Y D R O F U R A N O N B I O L O G I C A L SYSTEMS
325
TABLE 3. (Con’t)
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Exposure
Ref. Species Strain Sex Route
Duration
20. Rats
inhal. inhal.
3h 3h
M
i.p.
24 h
25. Guinea Pigs
NA
dermal single
2.
M inhal. &F
29.
NA
Guineapigs
Rats
NA
NA
Dose
0.29-0.59 mg/L 14.7mg/L
observation
- Only slight local irritation. - CNS irritation.
500mg/kg
- Normal serum omithine carbamyl transferase. No liver necrosis by H&E. Lipid accumulation under oil red stain. One of 4 died.
8.9 - 445 g/L
- No irritation or sensitization.
6 h/d; 13 wk 0.19-
14.7 mg/L
- 3 of 10 male mice died at 14.7 m a . Rats ataxic at 14.7 mgL. Narcosis in mice at 5.3 & 14.7 mg/L. Centrilobular hepatocytomegally in mice at 14.7 m a .
~~
~~~~~
(Abbr. used : NA - not available; SD - Sprague-Dawley; M - male; F - female; inhal. - inhalation.)
solvent.ll3 l2 The early data are summarized in a brief chapter by B r 0 ~ n i n g . lA~ more recent study, indicates a revived interest in the mammalian toxicity of THF? and THF has been selected for carcinogenicity bioassay sponsored by the National Toxicological Program.l4 Studies on the lethality of TI-F are summarized in Table 2. Most of the studies on the lethality of THF were conducted in the late 60s and early 70s. These
provide estimates for acute L D ~ o of s 2 - 3 g k g and 20 - 70 mg/L for oral and inhalation routes of exposure, respectively (Table 2). A single study using intravenous administration reported an LD50 of 759 rng/kg.l* Most of these studies employed rats and mice, with only slight species differences (Table 2). In three comparative studies, rats were consistently slightly less susceptable to the
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lethal effects of THF, and in one of these studies, guinea pigs were equivalent to mice.17. 19*2o THF was weakly toxic in two studies conducted with fish.21*22 In a single study on wild birds, feed containing THF was avoided, so no toxicity Drug and Chemical Toxicology Downloaded from informahealthcare.com by Nyu Medical Center on 11/09/14 For personal use only.
data could be determi11ed.2~In addition, in a single study in rats, only slight differences in toxicity due to age were noted.16 Based upon these, and similar studies, the OSHA air standard for THF is 200 ppm. It is interesting that in Russia in 1969, Pozdnyakova l9 recommended a similar threshold of 0.5 mgil (170 ppm). Based on oral toxicity studies, and employing 1/1000 of the dose giving fiist observable signs of toxicity, Kimura et af. l6 have recommended a suggested maximal permissible single oral dose of 2 Ivkg (i.e. 0.15 ml/75 kg). The clinical signs, and gross and histological changes associated with THF poisoning are summarized in Tables 3 and 4. In general, high doses of THF induce anesthesia with a narrow therapeutic index of 1.2.'* At high doses, nonspecific changes in kidney, thyroid, and liver cells have been reported, along with leukocytosis (Table 3). Following inhalation exposure more specific changes to lung epithelium have been observed. Atmospheric exposure to THF results in dermal, as well as inhalation routes of exposure. While several studies were conducted in this manner, little attention was paid to transdermal absorption. A singular inhalation study comments on irritation to the skin.a In contrast, the only study employing direct application to the skin noted no irritating or sensitizing effects of THF25 While not substantiated by reported experiments, it has been suggested that the variation in irritating qualities of THF are associated with the presence, or absence, of peroxides in the preparation used. Hepatotoxicity is one of the more consistent responses noted to acute and chronic THF exposure. This has generally been restricted to high dose or prolonged exposures. For example, a "focal proliferative response" was noted in
32 7
E F F E C T O F TETRAHYDROFURAN ON B I O L O G I C A L SYSTEMS
TABLE 4. Effect of Specific Functions Following Exposure of Animals to THF.
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Exposure Ref. Species Strain Sex Route Duration
Dose
Observation
11.
Rats
NA
NA
inhal.
1.5h
191 mg/L
- Some fatty change in liver
30.
Rabbits NA
NA
inhal.
4h
0.29 35.3 mg/L
- Dose dependent effects on ciliary function.
31.
Rats
NA
inhal.
4 Nd; 12 wk
0.59 & 2.94 mg/L
- Slight irritation of nasal &
NA
32.
Mice
NA
NA
7.
Rats
Wistar M
33.
Rats
NA
NA
tracheal epithelium (low dose) - Severe damage to nasal & tracheal epithelium,with decreased cilia, vacuohtion, and increased d e m granules (high dose).
dermal single
NA
- Increased leukocytes, little change to skin.
inhal.
6h/d; 2-18 wk
0.59 5.88 mg/L
- Accumulation of body burden first 2-8 wk, followed by dimunition at all doses. Decrease in liver alcohol and formaldehyde dehydrogenases at 18 wk. No significant effect on cerebellar or gluteal creatinine kinase, succinate dehydmgenase, or acetylcholineesterase.
inhal.
30min
44.1 mg/L
inhal.
30min/d 7d 30min 8-18 wk
44.1 m g 5
- Cerebellum norA decreased immediately,cerebrum DA increased after 48 h. - Cerebrum DA decreased after 48 h. - Increased cerebrum norA and DA.
inhal.
44.1 m g 5
(Abbreviations used : NA - not available; SD - Sprague-Dawley;M - male; F - female; inhal. inhalation; norA - noradrenaline; DA - dopamine.)
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rats inhaling 5.6 - 12.2
"necrotic foci" in mice and rats receiving lethal
doses l7 and, "some loss of liver function" in rats inhaling 2 pg/L for 3 months.26
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Three studies employing a range of doses provide a better set of data to consider the hepatic responses to THF.2.
273
28 In rats receiving THF in their drinking
water for 4 weeks at doses of 1, 10, 100, and 1000 mgJL (corresponding to intake
of 0.1 - 125 mg / kg / day), slight histological changes in liver cells were observed only at the highest dose.27 In one inhalation study with rats 0.29,0.59,2.94, and 14.7 mg THFL for 4 Wday for 12 weeks, liver function tests were affected at the 2 highest doses?* while in another study with rats and mice receiving doses of 0.19,0.59, 1.76,5.29, and 14.7 mg/L for 13 weeks, centrilobular hepatocytomegally was only noted in test animals receiving the highest dose.2 In a single study with guinea pigs,29 THF was given i.p. at a dose of 500 mglkg. While there was no increase in the serum level of a liver cell specific enzyme, omithine carbamyl transferase, lipid accumulation was noted in sections of liver stained with oil red 0. Specific changes in liver enzymes following in vivo and in virro exposure to THF have been described, and will be discussed in a later section. A number of additional studies have focused on changes in specific cellular functions or constituents produced by exposure to THF (Table 4). Of particular note, tracheal ciliary function is d e p r e ~ s e d and , ~ ~alterations in the CNS content of neurogenic amines has been noted following inhalation of relatively high doses of THF.
and Cvt-
of THF,
Direct application of THF to bacterial mutagenicity testing systems, with and without microsome activating systems added, have revealed no mutagenic activity
E F F E C T O F TETRAHYDROFURAN ON B I O L O G I C A L S Y S T E M S
329
of THF at concentrations up to 14 mM. Tests at higher concenuations have been hampered by the cytotoxic action of THF to the bacteria (Table 5). Exposure of guinea pig leukocytes, to 742 mM THF also demonstrated its cytotoxic effect,40 Drug and Chemical Toxicology Downloaded from informahealthcare.com by Nyu Medical Center on 11/09/14 For personal use only.
while exposure of ascites BP8 cells to 1 mM THF was without effect.39 THF was also negative in a model membrane-electrode system which binds reactive polycyclic h y d r o c a r b ~ n s .In~ ~a single initiation-promotion study for skin papillomas, the use of THF as the initiator resulted in fewer papillomas than the comparative solvent, acetone. Papillomas in mice initiated with either solvent, and receiving the full promotional regimen were far fewer than in mice initiated with any of the positive test c a r ~ i n o g e n s .The ~ ~ data presented in these papers provide no evidence for THF having a genotoxic effect in bacterial or mammalian cellsP1 The results of a planned carcinogenesis bioassay for THF l4 have not yet been published.
Many of the available studies on the effects of THF in bacterial-mutagenicity and tumor initiation-promotiontesting were primarily concerned with l"s solvent effect on the test system (Table 6). In 1981, Maron et al. 36 first compared the ability of a number of solvents to affect the Ame's assay test for mutagenicity using benzo(a)pyrene as the positive mutagen. While they did remark on the cytotoxicity of THF at concentrations exceeding 25 puplate, they did not find THF to have any influence on the rate of mutagenesis associated with benzo(a)pyrene. Shonly thereafter, Arimoto et al. 42 also compared the effects of solvents on the mutagenicity of uyptophan-pyrolysate mutagens. In this instance,
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TABLE 5. Genotoxicity Studies on THF
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Ref.
Test System
Activation System
Concentration
Observation
34.
D3052 TA1538 TA98 C3076 TA1537 G56 TA1535 TAlOO wp2 WP2uvrA-
-&+
1.4 to 13.9 mM
- All negative
35.
TAlOO TA1535 TA98 TAlOO TA1537
-&+ -&+
3 pmol/plale 0.03-3 pmovplate
- Negative - Negative
36.
TAlOO
0.31 - 6.2 poVplate
- No mutagenesis - Cytotoxic;25%
TA98
37.
_-
TA1535 TA1537
_-
Copper-phlhalocyanine membrane -electrode (model system for mutagen binding)
0.31 - 6.2 pol/plate
at 0.31; 67% at 0.62; and 100% at higher conc. - Similar cytotoxicity for these strains.
0.05 mM
- Negative
B. Other Svslerlls; 38.
Tumor initialing / F-SENCAR mice
in situ
0.2 ml (2.5 mmol)
- Incidenceof papillomas less then with acetone as solvent.
39.
Cell replication of Ascites BP8 cells
in situ
1 mM for 48 h
- No inhibition of growth.
40.
Survival of Guinea pig leukocytes
in situ
371 mM / 15 min 742 mM / 15 min
- 3% loss of cells - 66%loss of cells.
the use of THF as the solvent (as compared to DMSO)enhanced the mutagenicity of Trp-P-1 13-fold. Additional tests with ethanol and acetonitrile, also enhancers
of mutagenesis in the same study, suggested that the solvent effect was mutagendependent.
EFFECT OF TETRAHYDROFURAN O N BIOLOGICAL SYSTEMS
331
TABLE 6. Solvent Effects of THF
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Ref.
36. 42.
Test System
Concentration
Benzo(a)pyene mutagenicity to TAl00 (+S9)
25 - 50 u p l a t e
Trp-P-1 mutagenicity to TA98 (+S9)
25 @/plate
Observation
- No stimulationof mutagenesis, some cytotoxicity.
508~75&/plate
- 13-fold stimulation of mutagenicity - killing effect
43.
As solvent (relative to acetone) for skin tumor initiating capacity of: BenzoOpyrene (SP) NA - Acetone best solvent. BP-dihymdiol-9,10-epoxide NA - THF>benzene>acetoneas solvents.
38.
As solvent (relative to acetone) for skin tumor initiating capacity of: - Decreased tumor response 70% Dibenz(aj]anlhracene @BA) 0.2 ml on skin - Decreased tumor response 50% 0.2 ml on skin DBA-3,4-di0l DBA-3,4-diol-1,2-epoxide 0.2 ml on skin - Increased tumor response 130-200% - Decreased tumor response 80% Cholanthrene (Chol) 0.2 ml on skin - Increased tumor response 105% 0.2 ml on skin Chol-9,19-diOI - Increased tumor response 200% Chol-9,19-diol-7.8epoxide 0.2 ml on skin
-
-
44. As solvent for DNA adduct formation (peroxide vs. peroxide-free “HF) - Adducts directly formed by Michael Light protected Reaction of trans4 hydroxy- 2-nonenal (i.e. peroxide-free) addition Not light protested - Adducts formed indirectly from with deoxyguanosine (i.e. peroxide) epoxide generated from THF peroxide. 45 *
As solvent for Cyromazine pesticide, effect on: Adult emergence LDSO range from larvae Adult emergence from pupae
46.
LD50 range
- alone: LDSO = 15.9 pg/L - methanol: LD50 = 22.0 pg/L - THF: LD50 = 3.8 ClgR. - methanol: LDSO = 30.9 pg/L - THF:LD50 = 9.4 pg/L
As solvent (relative to ethanol) to deliver ionophore ETH 1001 - 50% the effectiveness of ethanol Promotion of calcium 4456 m a efflux from calcium loaded, inside-out red blood cells.
The solvent effect of THF has also been examined in the skin tumor initiationpromotion test system (Table 6), where, as mentioned above, THF was negative
as an initiator. In 1977, Slaga et al. 43 compared the initiating capability of the
MOODY
3 32
parent carcinogen, benzo(a)pyrene (BP), to its putative proximal metabolite
BP-9,10-dihydrodiol-9,1O-epoxide. When acetone was used as a solvent, the metabolite was only a weak initiator in comparison to the parent compound. With Drug and Chemical Toxicology Downloaded from informahealthcare.com by Nyu Medical Center on 11/09/14 For personal use only.
THF as the solvent, however, not only did the metabolite demonstrate initiating capability, but in this instance, it exceeded the parent carcinogen (Table 6). Sawyer et ~ l . conducted ? ~ a similar series of experiments comparing dibenz(aj)anthracene and cholanthrene, with their respective diol and diol-epoxide metabolites. Again, THF was found to enhance the absorbance of the highly reactive metabolites resulting in stimulation of their initiating capabilities. THF was also found to significantly enhance the penetration of the pesticide cyromazine when compared to application of the pesticide without solvent, or with methan01.4~In a somewhat different test system, THF,when compared to ethanol, diminished the effectiveness of an ionophore to induce release of calcium from membrane vesicles.& These reports suggest that THF can be effective in facilitating the absorbance of certain cytotoxins, particularly highly reactive epoxides.
In a study that considered the effectof THF as a solvent in an in virro test for genotoxicity, another aspect of THF formulations was revealed. Sodum and Chung 44 compared the adducts formed from mixture of trans-Chydroxy-2nonenal (tHN) with guanosine, using light protected versus light exposed THF as the solvent. They found that the THF preparation which was purposely handled to facilitate peroxide formation resulted in a completely different set of adducts, then when the peroxide-free formulation was used. From examination of the structures of the 2 sets of adducts, they concluded that THF-peroxides catalyzed the formation of an epoxide of tHN. Adducts formed in this solvent system
E F F E C T OF TETRAHYUROFURAN ON B I O L O G I C A L S Y S T E M S
333
therefore arose from prior formation of the tHN epoxide, while in the peroxide free solvent system tHN could form adducts by direct Michaels addition reactions.
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These studies point out the potential for peroxide containing THF to modify selective solutes.
Effect of THF on M i s a e o u s In Vitro S v m
The effect of THF on a number of biological systems has been examined (Table 7)47 THF, at 20 mM, was found to produce slight inhibition of soya meal urease,48 to inhibit phospholipase A2 with a Ki of 12.5 mM?' to activate components of the phosphorylase a to b conversion at concentrations ranging from 50 to 1000 m M > O to inhibit superoxide generation in suspended leukocytes?0 and have no effect on cholinergic receptors at motor endplates (direct micro application)?' or on ciliary activity of cultured tracheal epithelium at 5 mM.% While these studies demonstrate that THF can modulate the activity of a number of biological functions, they have not been carried out with enough thoroughness to evaluate the potential for THF to modulate these activities in intact animals. More thorough studies, however, have been carried out on the effect of
THF on drug-metabolizing enzymes.
The in vivo effect of THF on P450-dependent mixed function oxidase (MFO) activities are summarized in Table 8. Moderate induction of select MFO activities was noted in two studies in which THF was given either as a single i.p. injection
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TABLE 7. In Vitro Effects of THF on Miscellaneous Biological Systems ~~
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Ref.
Test System / Source
48. Effect on bacterial urease /
Concentration
Observation
20 mM
- Slight or no inhibition.
50 mM
- 3.4-fold activation at 1 min.
Soya meal
50.
Effect on phosphorylase A / Intack M-SD rat hepatocytes
200 mM
- 4.8-fold activation at 1 min.
49 *
Ki = 12.5 mM Effect on phosphorylase A2 / Pig pancreatic phopholipase hydrolysis
- Noncompetitive inhibition.
51.
Effect on cholinergic motor endplate ND receptors / Isolated sartorius muscle of frog (Rana pipiens)
- Single pulses delivered by electro-osmosis fully block ACh-potentials & deploarizing activity of butyrolactone.
52. Effect on glycogen phosphorylase
1M
- 18.6-fold activation at 5 min.
kinase /Rabbit skeletal muscle 53.
1.24 M Effect on AMP activation of glycogen phosphorylase / Rabbit skeletal muscle
- Inhibition to 16%of control.
40.
Effect on leukocyte superoxide generation /Guinea pig peritoneal leukocytes, stimulated by antibodyantigen complex
- Inhibition to 42% - Inhibition to 25% - Inhibition to 0% - Cell killing
54.
Effect on ciliary activity / 5 mM Chicken embryo trachea organ cultures
63mM 124 mM 248 mM 4968~744mM
- No effect up to 60 min.
(2.16 gkg)?5 or by inhalation for 18 weeks (0.6 - 5.9
respectively. No
effect was noted, however, in rats receiving 0.1 - 96 mg THF/kg in their drinking water for 4 weeks.27 These studies suggest that THF, at a sufficient dose, may be a selective inducer of MFO activities after single or prolonged treatment. A single i.p. injection of 100 mg THFfl
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THE EFFECT OF TETRAHYDROFURAN ON BIOLOGICAL SYSTEMS: DOES A HEPATOTOXIC POTENTIAL EXIST ?
David E. Moody Center for Human Toxicology, Department of Pharmacology and Toxicology, University of Utah College of Pharmacy, Salt Lake City, UT 84108
ABSTRACT Tetrahydrofuran (THF) is a widely used solvent in industry and research. THF is a weak toxin, with approximate acute L D ~ o in s the range of 2 to 3 gkg, 8 to 20 mg/L, and 800 mgkg following oral, inhalatlon, and i.v. routes, respectively. The two primary signs of intoxication are narcosis and hepatocellular dysfunction, both occurring at doses of approximately one-half of the lethal dose. Little is known concerning the pharmacokinetics of THF. No evidence exists for genotoxicity due to THF. Ongoing carcinogenicity bioassay tests sponsored by the National Toxicology Program have not yet been completed. Two significant interactions of THF with cellular components have been studied; first, THF inhibits a number of enzymatic reactions at concentrationsranging from 10 to 100 mM. Most notably, THF is an inhibitor of a number of cytochrome P450 (P450) dependent mixed function oxidase activities, with a particular affinity for the alcohol-inducedisozyme (P450IIEl). Second, THF has been noted to enhance the toxic action of a number of compounds (i.e. solvent effect), in particular stimulating the more rapid absorption of reactive metabolites. A third factor to be considered is THF's ability to form peroxides upon standing. Little is known concerning the toxicity of THF peroxides. Therefore, while there is little evidence to suggest that THF would be a direct (Type I) hepatotoxin at relatively low doses, its ability to inhibit drug-metabolizing reactions (perhaps more so in alcohol users), and enhance the absorbance of reactive metabolites, are relatively unexplored avenues for THF to contribute to a hepatotoxic response. As a great deal of the human hepatotoxicity (Type 11) is "idiosyncratic"and cannot be readily reproduced in experimental animals, this potential route of hepatotoxicity should be considered in future evaluations of human exposure to THF. 319 Copyright 0 1991 b y Marcel Dekker, Inc.
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320
I " Tetrahydrofuran (THF), a four carbon cyclic ether, is a colorless flammable Drug and Chemical Toxicology Downloaded from informahealthcare.com by Nyu Medical Center on 11/09/14 For personal use only.
liquid with a smell similar to acetone which is widely employed as a solvent in industrial, pharmaceutical and research settings. The solvent qualities of THF are characterized by its high solubility in water and its ability to complex solutes through hydrogen bonding.' A saturated, unsubstituted furan, THF is a relatively nonreactive ether, with the exception, that peroxides of THF will form upon exposure to oxygen and sunlight. Additional characteristics and general information about THF are summarized in Table 1. The largest use of THF is as an industrial solvent, primarily for dissolution of plastic resins and as a reaction medium. Approximately 150 million lbs. of THF
are produced annually in the U.S. resulting in an estimated exposure of 90,OOO workers.2 The industrial use of THF leads to potential human exposure, through both occupation and generation of industrial wastes. In regard to the latter, THF has been identified in samples from toxic waste sites,3 in drinking water? and in the milk of mothers living in industrial
environment^.^
In addition, THF is
approved as a pharmaceutical solvent, allowing exposure from residues or direct application. Direct application is becoming a greater potential, as THF is employed in novel drug delivery formulations. The purpose of the following report was to review the literature concerning the biological effects of THF. Particular attention was paid to the potential for THF to cause hepatotoxicity.
METHODS Literature citations were collected following Med-line and Tox-line searchs for articles related to the "Biological effects of THF on mammalian systems". These
E F F E C T O F TETRAHYDROFURAN ON B I O L O G I C A L SYSTEMS
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TABLE 1. General Information About Tetrahydmfuran (THF)
Synonyms
Furan, tetrah ydro-(Chemical Abstracts); Cyclo-tetramethyleneoxide; butane, 1,4-epoxy ;and butylene oxide
CAS #
109-99-9
RTECS #
Lu5950000
Molecular formula
C4H80
Molecular weight
72.1
Boiling point
65 - 66 O C
Vapour pressure
114 mm Hg at 15 O C
Specific gravity
0.891
Water solubility
30 g at 25 O C
Conversion factors
1 ppm = 2.94 mg/m3 1 mg/L = 340 ppm 1% (v/v) = 124 mM
Tetrahydmfuran WF)
citations were supplemented by abstracts listed in Chem Abstracts (1964 - 1989) under "Furan, tetrahydro- : biological effects". Literature was used if the entire copy of the paper was available at the local libraries, or an abstract was available and contained sufficient data in itself for evaluation.
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There is a dearth of knowledge concerning the p h m a c o - (toxico-) kinetics of
THF. A single study demonstrated that in rats exposed by inhalation, THF accumulated in the brain and fat tissue samples collected (other tissue were not studied), with saturation following 8 (lower doses) to 2 (high dose) weeks of exposure, and subsequent decreases in the body b ~ r d e n .This ~ study, within the realm of the current search, was the only data available on tissue levels of THF (i.e. not even blood levels are available) after any route of administration. Likewise, essentially no published experiments concerning the metabolism of THF exist. Fujita conducted a series of experiments on the tetrahydrofurfuryl mercaptan moiety which demonstrated the saturated furan ring can be cleaved by oxidative metabolism, and Boyd has reviewed a number of studies which consider the metabolism of unsaturated, substituted furans, which may, or may not, apply to the saturated, unsubstituted THF. Kawalek and Anders lo have shown that incubation of THF with liver 9000 x g supernatant from rats pretreated with Aroclor or phenobarbital does result in formaldehyde generation, a common product of mixed function oxidase reactions. The metabolites of THF in biological systems have not yet been elucidated.
The early studies on the biological effects of THF in the 1930s focused on its anesthetic activity, but also included one of the first LD50 determinations for this
E F F E C T O F TETRAHYDROFURAN ON B I O L O G I C A L SYSTEMS
323
TABLE 2. Lethality of THF
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Exposure Ref. Species ~
Strain
Sex
Dose
Route
Duration
inhal.
NA
LC64 LC50 LC16
12.2 mg/L 8.3 mgR. 5.6 m g L
oral
14 d
LD50
2.05 g k g 3.21 glkg 2.85 glkg
oral
single dose
inhal.
2h
LDlOO LDmin LDlOO LDmin LDlOO LDmin
5.0 glkg 3.0 glkg 56-103 m g L 28-55 mgjL 24-29 mg/L 18-21 mg/L
Parameter
~
15.
Rats
Albino
16.
Rats
SD
17.
Rats
NA
NA
M(16-50g) M (80-160 8) M (300-470 g) NA
Mice
NA
NA
inhal.
2h
12.
Mice
NA
NA
inhal.
2h
LD50
72 m a
18.
Mice
NA
NA
i.v.
2h
LD5O
759 mglkg
19.
NA Mice Rats Guinea Pigs
NA
oral
single dose
LD5O LD50 LD50
20.
Mice
NA
NA
LD50
2.50 glkg
Rats
NA
NA
i.p. (20% single dose in olive oil) i.p. (20% in saline) inhal. 3h
MLC
1.90 g k g 2.90 g/kg 61.8 mg/L
LC50
2460 rn@
No effect Min. effect
216 mgR. 367 mgR.
21.
Fish
Fathead minnows
in water
96 h
22.
Fish
Fathead minnows
inwater 33d post-fertilization
(Abbr. used: NA - not available; SD - Sprague-Dawley;M - male; F - female; inhal.- inhalation.)
TABLE 3. Gross and Histological Changes Associated with Exposure to THF ~~
Exposure Ref. Species Strain Sex Route Duration
Dose
Observation
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~~~
15.
Rats
Albino NA
17.
Mice
NA
19.
NA
Rats
NA
NA
Mice
NA
NA
inhal.
NA
5.6 - 12.2 mg/L
-Focal proliferative response in liver (like cirrhosis)
oral
acute
5&3gPcg
- Clinical symptoms within
inhal. inhal. inhal. inhal.
2h 24-29mgL 2h 56- 103mg/L 2h/d;2mt 6 - 8 m g / L 4 h/d;6mt 1 -2mg/L
oral
5mt
5&10mglkg
3-5 min.: somnolence, bradypnea, cyanosis; necrosis of gastric mucosa, enteritis; endemitis cerebri; hemorragic and necrotic foci in liver, kidneys, and spleen. - Similar to oral, but more pronounced lung changes. - Some decrease in body weight and slight histological changes. - Leukocytosis, depression of blood pressure and some proliferative changes in lung.
- No harmful effects at 5 m a g . Growth of mice inhibited at 10 mag.
Rats Rabbits inhal.
constant;3mt 2 u g L
26.
Rats
NA
NA
27.
Rats
SD
M drinking 28d & F water
1.10, loo,& l,OOOmg/L
- Some loss of liver function. - No clinical signs. - No weight loss. - No change in mixed
function oxidase activities. - At high dose, mild histological changes to thyroid, liver, and kidney tubule cells.
24.
Rats
NA
M
inhal. inhal. inhal.
single 44.1 mg/L 30 min/d; 7d 44.1 mg/L 60 min/d; 8 wk 8.8 mg/L
- Irritation, skin and mucous membranes in all groups. - Papillary hyperplasia of lung. - No changes in serum GOT, GPT & AP.
28.
Rats
NA
NA
inhal.
4 h/d; 12 wk 0.29 14.7 mgR.
- Mucousal irritation at all doses. Serum liver function tests altered at 2.9 mgn. Pulmonary mucousal, wbc counts, and liver function altered at 14.7 mg/L. (Con't)
EFFECT OF T E T R A H Y D R O F U R A N O N B I O L O G I C A L SYSTEMS
325
TABLE 3. (Con’t)
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Exposure
Ref. Species Strain Sex Route
Duration
20. Rats
inhal. inhal.
3h 3h
M
i.p.
24 h
25. Guinea Pigs
NA
dermal single
2.
M inhal. &F
29.
NA
Guineapigs
Rats
NA
NA
Dose
0.29-0.59 mg/L 14.7mg/L
observation
- Only slight local irritation. - CNS irritation.
500mg/kg
- Normal serum omithine carbamyl transferase. No liver necrosis by H&E. Lipid accumulation under oil red stain. One of 4 died.
8.9 - 445 g/L
- No irritation or sensitization.
6 h/d; 13 wk 0.19-
14.7 mg/L
- 3 of 10 male mice died at 14.7 m a . Rats ataxic at 14.7 mgL. Narcosis in mice at 5.3 & 14.7 mg/L. Centrilobular hepatocytomegally in mice at 14.7 m a .
~~
~~~~~
(Abbr. used : NA - not available; SD - Sprague-Dawley; M - male; F - female; inhal. - inhalation.)
solvent.ll3 l2 The early data are summarized in a brief chapter by B r 0 ~ n i n g . lA~ more recent study, indicates a revived interest in the mammalian toxicity of THF? and THF has been selected for carcinogenicity bioassay sponsored by the National Toxicological Program.l4 Studies on the lethality of TI-F are summarized in Table 2. Most of the studies on the lethality of THF were conducted in the late 60s and early 70s. These
provide estimates for acute L D ~ o of s 2 - 3 g k g and 20 - 70 mg/L for oral and inhalation routes of exposure, respectively (Table 2). A single study using intravenous administration reported an LD50 of 759 rng/kg.l* Most of these studies employed rats and mice, with only slight species differences (Table 2). In three comparative studies, rats were consistently slightly less susceptable to the
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lethal effects of THF, and in one of these studies, guinea pigs were equivalent to mice.17. 19*2o THF was weakly toxic in two studies conducted with fish.21*22 In a single study on wild birds, feed containing THF was avoided, so no toxicity Drug and Chemical Toxicology Downloaded from informahealthcare.com by Nyu Medical Center on 11/09/14 For personal use only.
data could be determi11ed.2~In addition, in a single study in rats, only slight differences in toxicity due to age were noted.16 Based upon these, and similar studies, the OSHA air standard for THF is 200 ppm. It is interesting that in Russia in 1969, Pozdnyakova l9 recommended a similar threshold of 0.5 mgil (170 ppm). Based on oral toxicity studies, and employing 1/1000 of the dose giving fiist observable signs of toxicity, Kimura et af. l6 have recommended a suggested maximal permissible single oral dose of 2 Ivkg (i.e. 0.15 ml/75 kg). The clinical signs, and gross and histological changes associated with THF poisoning are summarized in Tables 3 and 4. In general, high doses of THF induce anesthesia with a narrow therapeutic index of 1.2.'* At high doses, nonspecific changes in kidney, thyroid, and liver cells have been reported, along with leukocytosis (Table 3). Following inhalation exposure more specific changes to lung epithelium have been observed. Atmospheric exposure to THF results in dermal, as well as inhalation routes of exposure. While several studies were conducted in this manner, little attention was paid to transdermal absorption. A singular inhalation study comments on irritation to the skin.a In contrast, the only study employing direct application to the skin noted no irritating or sensitizing effects of THF25 While not substantiated by reported experiments, it has been suggested that the variation in irritating qualities of THF are associated with the presence, or absence, of peroxides in the preparation used. Hepatotoxicity is one of the more consistent responses noted to acute and chronic THF exposure. This has generally been restricted to high dose or prolonged exposures. For example, a "focal proliferative response" was noted in
32 7
E F F E C T O F TETRAHYDROFURAN ON B I O L O G I C A L SYSTEMS
TABLE 4. Effect of Specific Functions Following Exposure of Animals to THF.
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Exposure Ref. Species Strain Sex Route Duration
Dose
Observation
11.
Rats
NA
NA
inhal.
1.5h
191 mg/L
- Some fatty change in liver
30.
Rabbits NA
NA
inhal.
4h
0.29 35.3 mg/L
- Dose dependent effects on ciliary function.
31.
Rats
NA
inhal.
4 Nd; 12 wk
0.59 & 2.94 mg/L
- Slight irritation of nasal &
NA
32.
Mice
NA
NA
7.
Rats
Wistar M
33.
Rats
NA
NA
tracheal epithelium (low dose) - Severe damage to nasal & tracheal epithelium,with decreased cilia, vacuohtion, and increased d e m granules (high dose).
dermal single
NA
- Increased leukocytes, little change to skin.
inhal.
6h/d; 2-18 wk
0.59 5.88 mg/L
- Accumulation of body burden first 2-8 wk, followed by dimunition at all doses. Decrease in liver alcohol and formaldehyde dehydrogenases at 18 wk. No significant effect on cerebellar or gluteal creatinine kinase, succinate dehydmgenase, or acetylcholineesterase.
inhal.
30min
44.1 mg/L
inhal.
30min/d 7d 30min 8-18 wk
44.1 m g 5
- Cerebellum norA decreased immediately,cerebrum DA increased after 48 h. - Cerebrum DA decreased after 48 h. - Increased cerebrum norA and DA.
inhal.
44.1 m g 5
(Abbreviations used : NA - not available; SD - Sprague-Dawley;M - male; F - female; inhal. inhalation; norA - noradrenaline; DA - dopamine.)
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rats inhaling 5.6 - 12.2
"necrotic foci" in mice and rats receiving lethal
doses l7 and, "some loss of liver function" in rats inhaling 2 pg/L for 3 months.26
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Three studies employing a range of doses provide a better set of data to consider the hepatic responses to THF.2.
273
28 In rats receiving THF in their drinking
water for 4 weeks at doses of 1, 10, 100, and 1000 mgJL (corresponding to intake
of 0.1 - 125 mg / kg / day), slight histological changes in liver cells were observed only at the highest dose.27 In one inhalation study with rats 0.29,0.59,2.94, and 14.7 mg THFL for 4 Wday for 12 weeks, liver function tests were affected at the 2 highest doses?* while in another study with rats and mice receiving doses of 0.19,0.59, 1.76,5.29, and 14.7 mg/L for 13 weeks, centrilobular hepatocytomegally was only noted in test animals receiving the highest dose.2 In a single study with guinea pigs,29 THF was given i.p. at a dose of 500 mglkg. While there was no increase in the serum level of a liver cell specific enzyme, omithine carbamyl transferase, lipid accumulation was noted in sections of liver stained with oil red 0. Specific changes in liver enzymes following in vivo and in virro exposure to THF have been described, and will be discussed in a later section. A number of additional studies have focused on changes in specific cellular functions or constituents produced by exposure to THF (Table 4). Of particular note, tracheal ciliary function is d e p r e ~ s e d and , ~ ~alterations in the CNS content of neurogenic amines has been noted following inhalation of relatively high doses of THF.
and Cvt-
of THF,
Direct application of THF to bacterial mutagenicity testing systems, with and without microsome activating systems added, have revealed no mutagenic activity
E F F E C T O F TETRAHYDROFURAN ON B I O L O G I C A L S Y S T E M S
329
of THF at concentrations up to 14 mM. Tests at higher concenuations have been hampered by the cytotoxic action of THF to the bacteria (Table 5). Exposure of guinea pig leukocytes, to 742 mM THF also demonstrated its cytotoxic effect,40 Drug and Chemical Toxicology Downloaded from informahealthcare.com by Nyu Medical Center on 11/09/14 For personal use only.
while exposure of ascites BP8 cells to 1 mM THF was without effect.39 THF was also negative in a model membrane-electrode system which binds reactive polycyclic h y d r o c a r b ~ n s .In~ ~a single initiation-promotion study for skin papillomas, the use of THF as the initiator resulted in fewer papillomas than the comparative solvent, acetone. Papillomas in mice initiated with either solvent, and receiving the full promotional regimen were far fewer than in mice initiated with any of the positive test c a r ~ i n o g e n s .The ~ ~ data presented in these papers provide no evidence for THF having a genotoxic effect in bacterial or mammalian cellsP1 The results of a planned carcinogenesis bioassay for THF l4 have not yet been published.
Many of the available studies on the effects of THF in bacterial-mutagenicity and tumor initiation-promotiontesting were primarily concerned with l"s solvent effect on the test system (Table 6). In 1981, Maron et al. 36 first compared the ability of a number of solvents to affect the Ame's assay test for mutagenicity using benzo(a)pyrene as the positive mutagen. While they did remark on the cytotoxicity of THF at concentrations exceeding 25 puplate, they did not find THF to have any influence on the rate of mutagenesis associated with benzo(a)pyrene. Shonly thereafter, Arimoto et al. 42 also compared the effects of solvents on the mutagenicity of uyptophan-pyrolysate mutagens. In this instance,
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TABLE 5. Genotoxicity Studies on THF
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Ref.
Test System
Activation System
Concentration
Observation
34.
D3052 TA1538 TA98 C3076 TA1537 G56 TA1535 TAlOO wp2 WP2uvrA-
-&+
1.4 to 13.9 mM
- All negative
35.
TAlOO TA1535 TA98 TAlOO TA1537
-&+ -&+
3 pmol/plale 0.03-3 pmovplate
- Negative - Negative
36.
TAlOO
0.31 - 6.2 poVplate
- No mutagenesis - Cytotoxic;25%
TA98
37.
_-
TA1535 TA1537
_-
Copper-phlhalocyanine membrane -electrode (model system for mutagen binding)
0.31 - 6.2 pol/plate
at 0.31; 67% at 0.62; and 100% at higher conc. - Similar cytotoxicity for these strains.
0.05 mM
- Negative
B. Other Svslerlls; 38.
Tumor initialing / F-SENCAR mice
in situ
0.2 ml (2.5 mmol)
- Incidenceof papillomas less then with acetone as solvent.
39.
Cell replication of Ascites BP8 cells
in situ
1 mM for 48 h
- No inhibition of growth.
40.
Survival of Guinea pig leukocytes
in situ
371 mM / 15 min 742 mM / 15 min
- 3% loss of cells - 66%loss of cells.
the use of THF as the solvent (as compared to DMSO)enhanced the mutagenicity of Trp-P-1 13-fold. Additional tests with ethanol and acetonitrile, also enhancers
of mutagenesis in the same study, suggested that the solvent effect was mutagendependent.
EFFECT OF TETRAHYDROFURAN O N BIOLOGICAL SYSTEMS
331
TABLE 6. Solvent Effects of THF
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Ref.
36. 42.
Test System
Concentration
Benzo(a)pyene mutagenicity to TAl00 (+S9)
25 - 50 u p l a t e
Trp-P-1 mutagenicity to TA98 (+S9)
25 @/plate
Observation
- No stimulationof mutagenesis, some cytotoxicity.
508~75&/plate
- 13-fold stimulation of mutagenicity - killing effect
43.
As solvent (relative to acetone) for skin tumor initiating capacity of: BenzoOpyrene (SP) NA - Acetone best solvent. BP-dihymdiol-9,10-epoxide NA - THF>benzene>acetoneas solvents.
38.
As solvent (relative to acetone) for skin tumor initiating capacity of: - Decreased tumor response 70% Dibenz(aj]anlhracene @BA) 0.2 ml on skin - Decreased tumor response 50% 0.2 ml on skin DBA-3,4-di0l DBA-3,4-diol-1,2-epoxide 0.2 ml on skin - Increased tumor response 130-200% - Decreased tumor response 80% Cholanthrene (Chol) 0.2 ml on skin - Increased tumor response 105% 0.2 ml on skin Chol-9,19-diOI - Increased tumor response 200% Chol-9,19-diol-7.8epoxide 0.2 ml on skin
-
-
44. As solvent for DNA adduct formation (peroxide vs. peroxide-free “HF) - Adducts directly formed by Michael Light protected Reaction of trans4 hydroxy- 2-nonenal (i.e. peroxide-free) addition Not light protested - Adducts formed indirectly from with deoxyguanosine (i.e. peroxide) epoxide generated from THF peroxide. 45 *
As solvent for Cyromazine pesticide, effect on: Adult emergence LDSO range from larvae Adult emergence from pupae
46.
LD50 range
- alone: LDSO = 15.9 pg/L - methanol: LD50 = 22.0 pg/L - THF: LD50 = 3.8 ClgR. - methanol: LDSO = 30.9 pg/L - THF:LD50 = 9.4 pg/L
As solvent (relative to ethanol) to deliver ionophore ETH 1001 - 50% the effectiveness of ethanol Promotion of calcium 4456 m a efflux from calcium loaded, inside-out red blood cells.
The solvent effect of THF has also been examined in the skin tumor initiationpromotion test system (Table 6), where, as mentioned above, THF was negative
as an initiator. In 1977, Slaga et al. 43 compared the initiating capability of the
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parent carcinogen, benzo(a)pyrene (BP), to its putative proximal metabolite
BP-9,10-dihydrodiol-9,1O-epoxide. When acetone was used as a solvent, the metabolite was only a weak initiator in comparison to the parent compound. With Drug and Chemical Toxicology Downloaded from informahealthcare.com by Nyu Medical Center on 11/09/14 For personal use only.
THF as the solvent, however, not only did the metabolite demonstrate initiating capability, but in this instance, it exceeded the parent carcinogen (Table 6). Sawyer et ~ l . conducted ? ~ a similar series of experiments comparing dibenz(aj)anthracene and cholanthrene, with their respective diol and diol-epoxide metabolites. Again, THF was found to enhance the absorbance of the highly reactive metabolites resulting in stimulation of their initiating capabilities. THF was also found to significantly enhance the penetration of the pesticide cyromazine when compared to application of the pesticide without solvent, or with methan01.4~In a somewhat different test system, THF,when compared to ethanol, diminished the effectiveness of an ionophore to induce release of calcium from membrane vesicles.& These reports suggest that THF can be effective in facilitating the absorbance of certain cytotoxins, particularly highly reactive epoxides.
In a study that considered the effectof THF as a solvent in an in virro test for genotoxicity, another aspect of THF formulations was revealed. Sodum and Chung 44 compared the adducts formed from mixture of trans-Chydroxy-2nonenal (tHN) with guanosine, using light protected versus light exposed THF as the solvent. They found that the THF preparation which was purposely handled to facilitate peroxide formation resulted in a completely different set of adducts, then when the peroxide-free formulation was used. From examination of the structures of the 2 sets of adducts, they concluded that THF-peroxides catalyzed the formation of an epoxide of tHN. Adducts formed in this solvent system
E F F E C T OF TETRAHYUROFURAN ON B I O L O G I C A L S Y S T E M S
333
therefore arose from prior formation of the tHN epoxide, while in the peroxide free solvent system tHN could form adducts by direct Michaels addition reactions.
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These studies point out the potential for peroxide containing THF to modify selective solutes.
Effect of THF on M i s a e o u s In Vitro S v m
The effect of THF on a number of biological systems has been examined (Table 7)47 THF, at 20 mM, was found to produce slight inhibition of soya meal urease,48 to inhibit phospholipase A2 with a Ki of 12.5 mM?' to activate components of the phosphorylase a to b conversion at concentrations ranging from 50 to 1000 m M > O to inhibit superoxide generation in suspended leukocytes?0 and have no effect on cholinergic receptors at motor endplates (direct micro application)?' or on ciliary activity of cultured tracheal epithelium at 5 mM.% While these studies demonstrate that THF can modulate the activity of a number of biological functions, they have not been carried out with enough thoroughness to evaluate the potential for THF to modulate these activities in intact animals. More thorough studies, however, have been carried out on the effect of
THF on drug-metabolizing enzymes.
The in vivo effect of THF on P450-dependent mixed function oxidase (MFO) activities are summarized in Table 8. Moderate induction of select MFO activities was noted in two studies in which THF was given either as a single i.p. injection
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TABLE 7. In Vitro Effects of THF on Miscellaneous Biological Systems ~~
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Ref.
Test System / Source
48. Effect on bacterial urease /
Concentration
Observation
20 mM
- Slight or no inhibition.
50 mM
- 3.4-fold activation at 1 min.
Soya meal
50.
Effect on phosphorylase A / Intack M-SD rat hepatocytes
200 mM
- 4.8-fold activation at 1 min.
49 *
Ki = 12.5 mM Effect on phosphorylase A2 / Pig pancreatic phopholipase hydrolysis
- Noncompetitive inhibition.
51.
Effect on cholinergic motor endplate ND receptors / Isolated sartorius muscle of frog (Rana pipiens)
- Single pulses delivered by electro-osmosis fully block ACh-potentials & deploarizing activity of butyrolactone.
52. Effect on glycogen phosphorylase
1M
- 18.6-fold activation at 5 min.
kinase /Rabbit skeletal muscle 53.
1.24 M Effect on AMP activation of glycogen phosphorylase / Rabbit skeletal muscle
- Inhibition to 16%of control.
40.
Effect on leukocyte superoxide generation /Guinea pig peritoneal leukocytes, stimulated by antibodyantigen complex
- Inhibition to 42% - Inhibition to 25% - Inhibition to 0% - Cell killing
54.
Effect on ciliary activity / 5 mM Chicken embryo trachea organ cultures
63mM 124 mM 248 mM 4968~744mM
- No effect up to 60 min.
(2.16 gkg)?5 or by inhalation for 18 weeks (0.6 - 5.9
respectively. No
effect was noted, however, in rats receiving 0.1 - 96 mg THF/kg in their drinking water for 4 weeks.27 These studies suggest that THF, at a sufficient dose, may be a selective inducer of MFO activities after single or prolonged treatment. A single i.p. injection of 100 mg THFfl
