Document KGY5EMraJK3DMyOzNJeXR1R9r

CudBogenMfr VoU No.B pp.1031 -1038. 1983 Vinylidene chloride; changes in drug-metabolizing enzymes, mutagenicity and relation to its targets for carcinogenesis F.Oesch1, M.Protit-Sablji6u, T.Friedberg1, H.-J.KUraisch1 and H.R.GIaR1 institute of Pharmacology, Obere Zahlbacher Strasse 67, D-6500 Mainz, and 3Department of Toxicology, BASF Aktiengesellschaft, D-6700 Ludwigshafen, FRG. (Received on 22 March 1983; accepted on 15 June 1983) Abstract Results of various sfcx&es have shown that male Swiss Webster mice are more susceptible to toxic effects of vinyl idene chloride (VDC) than are females of the same mouse strain, females and males of the C57BL mouse strain, Chinese hamsters and rats. The main targets of toxicity are kidney and liver. The kidney of male Swiss Webster mice is the only organ whereVpc unambiguously hiduces tumours, in tne present study we have investigated the aDmly"of NADPH-Iorltiled pottmitochoodrial supernatant fractions (S-9 mix) of kidney and liver from susceptible and nun susceptible enimaM to activate VDC to a bacterial mutagen. The foBowtng sequence of activating potencies was observed: mouse liver (both strains ana sexea) ana Chinese hamster liver > rai' Bver ? HflHiaw HVSf s lakeae namster kidney > kidney from mate mice 01 noth strains > kidney from rats aBdTiBBag hliCc. Ihe last two preparation. <%niv AwuetmiMiv fflWed weak acuvauon of VTxL Addition of nitrified microsonUB epoxide nydrolue to S-9 mix (fid not affect the muta genicity of VDC; addition of glutathione reduced the mutagenidty up to 50^o. Pretreatment of animals (male rats, male and female Swiss Webster mice) with VDC did not potentiate the ability of the subceUulsr preparations to activate this com pound. In fact, In some cases, a weaker activation was observed. Following this treatment, microsomal 7-ethoxycoumarin O-dealkylase was decreased in mouse kidney and in rat Kver. The enzyme was not affected in mouse liver and was not measurable in rat kidney. Microsomal epoxide hydrolase activity (with styrene 73-oxide as substrate) was not affected in mouse liver and rat kidney. In the kidney of male mice treated with a high concentration of VDC, epoxide hydrolase activity was decreased initially, hot after longer treatment. In some cases a weak: Increase above control was noticed. A stronger increase In activity of epoxide hydrolase was observ ed in the rat liver and the kidney of female mice. Cytosolic glutathione transferase activity (with 2,4-dlnltrochlorobenzene as substrate) was not affected by the VDC treatment In the liver of male mice, bat was decreased in the kidney of male mice, and was elevated la the kidney and liver of rats and of female mice. The different effects of VDC on titb efP zyme may be one of the reasons for the differences In suscep tibility towards the toxic and carcinogenic actions of this compound In different spedes, strains and sexes. *Present address: Laboratory of Molecular Carcinogenesi*. National Cancer Institute, Bethesda. MD 20203, USA. Permanent address: Laboratory for Marine Molecular Biology, Institute Rudjcr BofkovHf, Zagreb, Jugoslavia. *AbbrevfcMioa: VDC, vinylidene chicride. IRL Press Ltd.. Oxford, England. Introduction Vinylidene chloride (1,1-dichkwoethylene, VDC*), a reac tive chemical substance which is widely used in the produc tion of various copolymers, shows remarkable differences in toxicity in animals depending on species, strain, sex and nutri tional state (1--4). For instance, the acute inhalation toxicity is characterized by LC values which range from < 50 p.p.m. in fasting male mice to > 10 000 p.p.m. in fed female rats. The main targets of acute and chronic toxicity are the kidney and the liver. In addition to toxicity studies, a total of 15 car cinogenicity studies have been performed (2--12). In inhala tion experiments with Chinese hamsters, various strains of rats and female mice and in ingestion experiments with rats and mice, either completely negative results were obtained or a few sporadic tumours were found at various sites but they could not be clearly attributedto the action of VDC. The only substance-related tumorigenic effect was observed in male Swiss Webster mice, which developed adenocarcinomas in kidneys after inhalation of VDC (4,5). This effect occurred at exposure to 25 p.pjn. but not to 10 p.p.m. VDC. At 50 p.p.m., mortality was so high that an experiment could not be conducted. Hence, the carcinogenicity of VDC appears rather specific with respect to species, sex, organ and dose. Further more, the occurrence of an effect only dose to lethal doses and only in animals which are highly susceptible to toxic ef fects, suggests that there may exist a relationship between toxidty and cardnogooidty, e.g. a common active metabolite responsible for both effects or promotion of the tumour development by toxidty. An Important question arising from these animal data is whether or not VDC can induce tumours in man. Epidemio logical studies did not show any correlation between tumour incidences in workers and exposure to VDC during produc tion processes (13--15). An understanding of mechanisms underlying the highly selective carcinogenicity in animals would be a desirable rational complement to the negative em pirical carcinogenidty data in man. In studies with bacteria and yeast, VDC was not mutagenic itself (16--20). However, It was activated to a mutagen by subcellalar tissue preparations (16--20). Preparations from tire tumour-susceptible spedes, the mouse, were more effec tive than from rats (albeit not the same mouse strain as in the positive cardnogeniaty study was used) (17--19). Prepara tions from the target organ, kidney, showed much less activa tion of VDC to a mutagen titan liver preparations (17,19). However, it should be noted that these studies did not take in to account potential strain differences in cardnogmidty which may be expected from the strain differences in toxicity (2,4,12) - and potential changes in the metabolism due to enzyme induction during long-term treatment ofanimals with VDC. In order to Increase our understanding of events associated with these spedes, strain, and sex differences, we have now the effect of VDC treatment on several drug- metabotizing enzymes in the liver and kidney of tumoursusceptible male Swiss Webster mice and of non-susceptible 1031 AP00009950 F.Orach et al. female Swiss Webster mice and Sprague Dawley rats. The en zyme activities studied were 7-ethoxycoumarin O-dealkyiase, microsomal epoxide hydrolase with styrene oxide as substrate and glutathione transferase with 2,4-dinitrochlorobenzene as substrate. The same enzymes are potentially involved in the metabolism of VDC (21). We also studied the activation of VDC to a bacterial mutagen in parallel. Mutagenicity ex periments with metabolizing systems from non-treated animals of some other species and strains which have been us ed for carcinogenicity and toxicity experiments and from man were included for comparison. Materials and Methods CHemkets VDC (gas chromatographic purity: 99.996V* VDC; 27 p.p.m. methylchloride * virylchioride + chtaroacetylene; 6 p.pjn. dkhloroacetyicne; 4 p.p.m. tra-I.2^ichloroethyleie), stibifized with 200 p.p.m. 4-methoxyphenol (monomeihyl ether of hydroquinotie), and 4-methoxyphenol were ob tained from BASF AC, Ludwigaha/en, FRG. Animals Swiss Webster mice and Sprague Dawley rats were generously provided by Prof. C.Mtltoni, Bologna, and bred at BASF AC. Chinese hamster* Fue: FUST (bra) were from the same breeding (BASF AG) as used by Prof. CJvfaltoni (9). C57BL/6J Kan mice were purchased from the ZentmUnstitut far Versuchstlere, Hannowr, PRO. The male mice used in the induction ex periments weighed 30- 39 g, the females 27 -32 g and the rats 147-179 g. Treatment with VDC was performed in an inhalation chamber with a daily exposure for 6 h. The 8 day-treatmoit group was exposed to VDC on 8 se quential days, whereas diecontrol animal* were etposed to air. The 1 day-end 3 day-nearaent groups wee treatedwhh VDC on the last day or on the last 3 days of this 8 day-period and received sham treatments with air during the preceding days. The were killed with COj one day after the last ex posure. Livos and kidneys were removed under sterile conditions and homogenised using a glass-teflon homogenize* in cold, isotonic KC1 solution, which to rnM sodium phosphate buffer pH 7.4. The horaogmata were centrifuged for 10 min at 9000 g. A part of the resulting postmito- chondrlai supernatant fraction was (hen centrifuged for 60 qua at 100 000 g. The 100 000 g supernatant was used for detomlnation of glutathione transferase activity and the microsomal pellet was resuspended in the homogenization buffer and used for detominadon of epoxide hydrolase and 7-ethoxycoumarin O-dealkytMe activities. Enzyme assays 7-Eihoxywumarin O-dealkytase activity was measured by the method of UUrlch and Weber C22) at 37*C. The activity of epoxide hydrolase was deter mined by the method of Oesch <f al. (23) but without Tween 80 (24) with styrene oxide u substrate at 37BC. GhitatMooe transferase activity was measured by the method of Habig tt of. (23) with 2Adinitxociikxobenaene as substrata at 30C. All enzyme assays wvechecked for Hnearitywith respect to amount of protein and time foreach strain of animals. Protdn concentrations were measured by the method of Lowry tt at (26) using bovine wum Ihnm<n y Standard. Muiogenkity optrimenU The Air- 5Ubwiaite0'pAimmSumstrdmTA1335,TAI337,TA96,TA100 and TA92 were obtained from DrJLN.Anra, Berkeley, CA. The trp~ strain ExfwieHIa call WP2 uwA was a gifLof Dr.R.McMahan, Lilly Rrararcfa laboratories, Indianapofis, IN. Stock outturnwraestored at -70"C until used. The bacteria were grown overnight in Baao nutrient broth (8 g/1) which was supplemented with 3 g/1 NaO. Befbre the cxpcrhnmt, bacteria werecmtrifuged and resuspended to titer of --1.7 x 10* bacteria (colony forming unks) per ml In resuipenrion medium contained 1.6 g Bacto nutrient broth and 5 g NaO per Ikra. A mwaboHring synn, called S-9 mix, consisted of the following inedknts: 0) poauratodiondriai supernatant fraction from 6.23 to 200 mg tissue made up to a volume of 167 or 333 *d (depwdini on the experiment) with the buffer used for homogenization: (U) 167 of e solution containing 24 mM MgQi and 100 mM KC1; (111) 167 *1 ofa solution containing 12 mM NADP, IS mM glucose6-phosphate and 130 mM sodium phosphate buffer pH 7.4 and (iv) 100 ftl of aa appropriate solution ofglutathione or epoxidehydrolase (27) In 10 mM *<<" phosphate buffer adjusted to pH 7.4 or buffs only (this component was added only la experiments where glutathione or epoidde hydrolase was used). 1032 r The mutagenicity experiments with his~ S. typhinturium were performed with minor modification of the methods described by Ames tt al. (28) and. in eaae of the volatile VDC, Bartsch at al. (17). Experiments with trp~ E. coil were conducted analogously. A volume (300--767 ^i) of S-9 mix (or 500 130 mM KG), 100 jtl of the bacterial suspension and 2000^1 top agar (which consisted of 0.55V* agar, 0.55V* NaCl, 50 histidine, 50 pM biotin, 50 tryptophan and 25 mM sodium phosphate buffer pH 7.4, 4S*C) were mixed in a test tube and poured onto a Petri dish with minimal agar r^nd^ing of 1.54b agarand Vogd-Bonnw E medium with 2V* glucose. In the case of solid test compounds, the mixture also contained 10--30 /d of a solution of the compound in dimethyl sulfoxide. In the case of VDC. the plates were placed for 4 h in a desiccator with VDC. After a further incubation for 2-3 days In the absence of VDC it 37C in the dark, the colonies (ha * orop* revertanu) were counted. Results Mutagenicity in various bacterial strains Table I shows that metabolically activated VDC was mutagenic in all six bacterial strains used: 5. typhimurium TA1335, TA100 and TA92, three DNA repair variant strains derived from the substitution mutant his G46; S. typhi murium TA98 and TA1537, two different frameshift mutants; and E. coli WP2 uvrA, which possesses a substitu tion mutation in a gene required for tryptophan synthesis. The stabilizer 4-methoxyphenol could not account for any of these mutations, as it did not show mutagenic effects (Table I) . In the absence of an activating system, VDC and 4-methoxyphenol were not mutagenic (data not shown). Activation of VDC by various metabolizing systems The liver and the kidney are the main target organs for the toxic and carcinogenic effects of VDC. SubceUular prepara tions of these organs from various mammalian species were tested for their ability to activate VDC to a mutagen (Table II) . Liver S-9 mix from Chinese hamsters and mice were most active. Preparations from male Swiss Webster mice did not differ appreciably from preparations from females of the same strain nor from preparations from C57BL females. Preparations from C57BL males were also similarly active when used in low amount (12.5 mg tissue equivalents/plate). whilst they were less active than those from Swiss Webster mice when used in higher amounts (25 and 50 mg tissue equivalents/plate). Rat liver S-9 mix caused a clearly weaker activation than that from Swiss Webster mouse (see also Table V). Two preparations from human liver were also used. Both had similar activity. When large amounts of postmitochondrial fraction were used, they led to marked mutagenic effects, whereas at smaller concentrations they were much less active than liver preparations from other species. Preparations from kidney were substantially less active than those from liver. Preparations from Chinese hamsters and from male mice of both strains regularly led to a weak ac tivation, whereas those from female mice and from rats were mostly inactive (see also Table V). In a few experiments, kidney S-9 mix from rats and female mice weakly activated VDC. The reasons for the discrepancies among experiments are not dear, but may, at least in part, be due to the weakness of the mutagenicity. To investigate the possible roles of microsomal epoxide hydrolase and conjugation with glutathione in the activation and inactivation of VDC, we added epoxide hydrolase (purified from rat liver rakrosomes (XT)) or glutathione to S-9 mix from liver or kidney ofSwiss Webster mice. Fifty units of enzyme, a >20-fold excess over the endogenous activity in the S-9 mix, did not affect the mutagenicity of VDC (data not shown). The epoxide hydrolase inhibitor, 1,1,1-trichloro- AP00009951 YlayUdene chloride: enzyme tadwsioe iml muUgttiidty Table I. Investigation for liver enzyme-mediated mutagenicity and toxicity of VDC and of its stabilizer 4-methoxyphenoi in various bscteriai strains. Ten compound Surviving Fraction D p.p.m. VDC 375 2230 4300 1O5O0 22500 100 mg 4-Methoxyphenol per 201 desiccator 0 3.15 -1000 Ml 4-Methoxyphenol per place 1.0 1.1 1.0 1.0 1.0 1.0 1.0 1.0 0.9- 1.0 Revertant colonies/plate TA100 TA1S35 105 740 380 1270 1080 1380 125 102 93-103 24 223 490 300 550 660 22 9 8-n TA92 30 245 319 283 295 263 40 28 25-32 TA1537 17 42 43 52 41 46 25 17 16-25 TA98 32 202 247 m 274 314 39 36 34-39 WP2 uvrA 26 $60 600 690 740 690 23 21 20-25 Minimal agar plates with his~ S. typhimurium or trp~ . eoH strains and with NADPH-fortlfied liver postmitodtondrial supernatant fraction (corresponding to SO mgiiisue/plate) from untreated male adult Swiss Webster cniee were kept at 37*C for 4 hiu a desiccator with VDC. After further incubation for2 days at 37`C, colonies (revertanu) were counted. The stabilizer 4-methoxyphenol was tested by the same method (whereby no significant subHmadon in the desiccator was observed) or by adding it (at ^different concentrations) directly to the plates. The values are means from 2 to4 replicate incubations. The individual values deviated by < 109 from the mean values, except in scene cases where the numbers of colonies were low. Toxicity was estimated by determining the surviving fraction of 600 his* mutants which were added as an internal standard to mutagenicity plates (with TA1537). Table Q. Activation of VDC to a muiagsi by liver and kidney postmitochondrial supernatant fractions from various species. Experiment I n in [V V VI vu Organ Spedes Liver Liver Liver Kidney Kidney Swiss Webster mouse C57BL mouse Chinese hamster Sprague Dawley rat Human Human Swiss Webster mouse Swiss Webster mouse Sprague Dawley rai Chinese hamster C57BL mouse Kidney C57BL mouse Kidney X . CJ7BL mouse - 7: Sex mala female male female male male male male male male male male male male female .. <* p,p4B. VDC in mutagenicity experiments 4500 4300 4500 4300 4500 4300 2250 4900 2250 2250 4500 4500 4500 450 1250 4500 12500 90 450 1250 90 450 1250 45Q0 -- Number of induced revertants/olate Postmitodwndrfa] fraction (tissue eauivaientsl 6.25 mg 12.S mg 25 mg 50 mg 100 mg _fc 399 688 - 384 683 - 381 215 - 362 596 - 312 619 - 162 243 22 87 142 41 72 153 4 77 152 133 310 354 - 93 19 -- 171 _A -- --* --1 - _* -- _s -* - -- --1 _ --* _g -* - 11 --* 13 -- 18 6 7 -- 5 6 -* ' 10 613 719 296 370 654 345 278 305 345 473 67 41 105 126 106 106 139 31 70 113 7 3 -3 -12 A , - --* ' -* * _ . _ $84 529 60S 559 85 15 158 --1 - - -* -- - --* - -* - Hbtkfine-poor agarpilUe^coQtainftif tiis~ S. OpMnurfumTAlOQandNADPH-fortifted postmitochoodrial fraction from 6.25 to 100 mg liver or kidrvey were ex posed to VDC for<flL*t^*C.After further incubation for2daysat 37*C, his* revertant colonies were counted. Values aremeans from 2 (Experiment II, V, VI, VIJ) oc 3 (other experiments) replicgfe hKubatkm minus the conspondim number of tnntanti In the absence of VDC 198--133). The variation coefficients of the number of colonies onjtepllcate plates were <10fe.Tbe two human liversample* wentakanduring surgical treatment, transported on iceand used tor mutagenid- ty experiment! withtfSn aher they had been takoi. "Not determined. propene 2,3-oxide(29), also had no effect on the activation of VDC by S-9 mix (this experiment was performed with TA98 in presence of 1 mM tricWoropropene oxide; data not shown). Glutathione usually reduced the mutagenicity of VDC by between 20 and 507s. Typical examples are shown in Figure 1. However, glutathione increased the mutagenicity of VDC in some experiments, especially when weakly active preparations from kidney or when small amounts of liver preparations were used (data not shown). Kidney S-9 mix from female mice regularly activated VDC to a mutagen in the presence of glutathione, whereas this was usually not the case in its absence. The effect was always weak, maximally 100 colonies above solvent controls of -150 colonies. Experiments on the stability of kidney ami Aver 5-9 mix The weak activation of VDC by kidney preparations in comparison to liver preparations was somewhat surprising. Because extrahepatic subcellular fractions are rarely used for activation in mutagenicity experiments, little is known about their optimal preparation and their stability. Kidney prepara- 1033 AP00009952 F.Oesch tf at. 05 Glutathione ImM) 10 ng. i. Effect of glutathione on liver enrymo-mtdiaied mutagenicity of VDC. Minimal agar plates with hatidine-auxotrophic 5. typhimurtum TA100, NADPH-fortified, dialyzed liver pontmhochondrial supernatant frac tion and various concentrations ofglutathione were placed for 4 h at 37*C in a desiccator with0 p.pjn. (data not shown), 430 p,pjn. (upper panel) or4300 p.p.m. VDC (lower panel). Alts funhs Incubation for 2 days at 37*C, col onics (rcvetUuus) woe eountad. Valuee are means from 3 replicate plane. The variation coefficients were <10%. The number of spontaneous revertants varied from 88 to 133 (depending on amount of postmitochondrial fracdon and glutathione) and was not subtracted. Dialysis of the poctmltocfcoodrtal fraction removed >95% of die endogenous glutathione. The concentration of glutathione in the figure is related to the top agar layer. , pestmito chondrial fraction from 30 mg liver of untreeted male C57BL mice; , poet* nutochondrinl fraction from 30 mg Kw of untreated male Swiss Webster mice; O, pactmitochondriai friction from 100 rag liver of untreated male Swiss Webster mice. tions used in the present study activated 2-aminoanthracene and benao[a1pyrene (Table III and data not shown). The ac tivation of 2-aminoanthracene by kidney S-9 mix was even greater than by liver S-9 mix. It has been observed that ethylene, vinyl chloride and some other olefins after metabolic activation destroy cytochrome P-450 (30,31), the enzyme probably required for metabolic activation of VDC. However, rapid inactivation of kidney preparations when they are Wept at 37C or exposed to VDC in mutagenicity experiments is unlikely, because kidney and liver S-9 mix exposed to VDC before a mutagenicity experi ment maintained their ability to activate 2-aminoanthracene and VDC (Table III). Activities of drug-metabolizing enzymes in VDC-treated animals and effects on the actfvatkm of VDC Bartsch et al. (17) and Jones and Hathway (19) have demonstrated that enzyme induction by phenobarbital or Axoclor 1254 potentiates the activation of VDC by suboeUular preparations. Therefore, possible enzyme inducing effects of VDC itself may be of relevance in carcinogenicity experiments. Female mice and male rats were treated as described in previous carcinogenicity studies (4,5), Le. they were exposed daily for 6 h to 50 and 200 p.p.m. VDC, respec tively. Male mice were treated with 50 p.p.m., a concentra tion which was lethal for many animals, or with 10 p.pjn., a concentration at which all animals survived. The effects on enzyme activities of these treatments after various periods are 1034 Tabic m. Exposure of S-9 mix to VDC before the mutagenicity experiment: effect on the ability to activate prernutagens. Source and treatment of S-9 mix Number of Air* revotaius/nlate Control plates Plate* exposed Plates with to 4500 p.p.m. 1 mS 2-amlno* VDC anthracene Liver S-9 mix exposed to VDC at 37*C kept at 37*C kept at 0*C Kidney S-9 mix exposed to VDC at 37*C kept at 37*C kept at 0*C 142 10 12S * 3 120 * 15 205 * 24 132 11 151 * 2 900 90 1760 * 80 930 30 2S7 =fe 14 196 * 16 201 14 7800 400 5400 * 400 4400 300 8300 700 7000 * 300 8700 * 900 NADPH-fortified portmitochondrial supernatant fractions from organs of untreated male Swiss Websta mice were divided into 3 aliquots. The first ali quot was exposed in a large sealed glass bottle to 9000 p.p.m. VDC at 37*C fev 1 h. Meanwhile, the othv two aliquots were kept at 37*C and 0*0. respectively. Afterwards, aliquots equivalent to 30 mg tissue together with his~ S. typhimurium TAI00 were poured on minimal agar plates. Some plates also received the premutagea Z^minoanthracene. Others were placed for 4 h at 37*C Into a desiccator with VDC. Afts a further Incubation for 2 days at 37*C, Air4 revenant colonies wm counted. Values are means and S.D. from 3 replicate incubations. shown in detail in Table IV and are summarized in Table VI. In the mouse liver, changes in enzyme activity were always very small and may be readily explained by statistical disper sion. However, the weak increase in glutathione transferase activity in female mice after the longest treatment period may be real, since the kidney ofthe same animals and the Uver and the kidney of VDC-treated rats also showed an increase in this enzyme activity. In the rat liver, monooxygenase activity was decreased after the first treatment to less than half of that in control animals and remained low during the observation period, whereas epoxide hydrolase and glutathione transferase activities gradually increased to about 2- and 1.5-fold control activity, respectively. In the kidneys of mice of both sexes, monooxygenase ac tivity was decreased in animals treated for 1 day with 50 p.p.m. or for 3 days with 10 p.pjn. With both regimens, the activity was below the detection Hmh after 8 days of treat ment excqpt in one experiment (Experiment number 5 in Table IV) with male mice treated with 50 p.pjn. hi this aber rant experiment, monooxygenasc activity On control and treated animals) was above the usual control values and a relatively high mortality rate occurred in the longest treatment group. The effects of VDC treatment on renal epoxide hydrolase activity considerably varied among repeat experi ment. It appears that two antagonistic processes, a decrease and an increase in activity, competed. An early fall followed by a rise In activity was observed in single experiments in which males and female mice, respectively, were treated with 50 p.p.m. In the repeat experiment with males (with a higher mortality rate than in the first experiment) only the decrease in activity was observed, whereas in the other experiment with females (Experiment number 6 in Table IV) only a pro nounced increase was seen. Males exposed to the low VDC concentration showed a weak increase after the 8-day treat ment, but no initial decrease in epoxide hydrolase activity. Renal glutathione transferase activity was not markedly altered in male mice treated with the low concentration of VDC, was decreased to less than half of the control activity AP00009953 VtaytMeoc chlorides carym* tadedei and imilmnhllj Table IV. Effect of VDC treatment on drug-metabolizing enryma in mouse end rat. Experiment number, animals and treatment 1. Male Swiss Webster mice Control 10 p.p.m., 1 day 10 p.p.m., 3 days 10 p.p.m., 8 days 2. Male Swiss Webster mice Control 10 p.p.m., ] day 10 p.p.m., 3 days 10 p.p.m., 8 days 3. Mak Swiss Webster mice Control 50 p.p.m., 1 day SO p.p.m.t 3 days 50 p.pjn.t 8 days Number of treated and surviving animals Enzyme activities fnmcl product/min/mg protein) Liver Monooxygenase Epoxide hydrolase Glutathione transferase Kidney Mottooxygenase 5/5 5/5 5/5 5/5 25/25 25/25 25/25 25/25 5/5 10/10 10/8 10/7 1.27 * 0.24 1.12 * 0.13 1.54*0.34 1.52 * 0.17 1.69 1.16 1.19 1.09 1.19 * 0.30 1.44 0.12 1J0 * 0.46 1.42 * 0.18 123 * 0.39 1.98 * 0.18 2.41 6 0.51 2.53 * 0.29 3150 * 360 3390 490 3520 1080 2920 * 440 1.74 3980 1.59 4580 1.67 4180 1.50 4020 129 * 0.43 1S8 0.27 2.45 * 0.06 121 * 0.52 3360 * 2980 * 2890 * 2760 * 410 340 500 550 0.05 0.04 <0.01 <0.01 Epoxide hydrolase a 14 0.15 0.16 0.24 Glutathione transferase 441 314 359 427 4. Male Swiss Webster mice Control 25/25 50 p.p.ra., 1 day 50 p.pJiL, 3 days SO/SO 50/16 50 p.p.m.. 8 days J. Male Swim Webster mice 30/17 Control 50 p.p.m., 1 day 25/25 30/30 SO p.p;re., 3 days 35/16 50 p.pjn., 8 days 40/7 6. Female Swiss Webster mice Control 10/10 50 p.p.in,, 1 day 15/15 50 p.p.ra^ 3 days 50 p.p.m., 8 days 15/15 15/15 7. Female Swiss Webster mice Control 50 p.p.m., 1 day 50 p.p.m., 3 days 25/25 25/25 25/25 SO p.p.m., 8 days a Male Sprague Dawley* nus Control 25/25 10/10 200 p.pja.t I day 200 p.p.m., 3 day* 10/10 10/10 200 p.p.Bu* 8 days 9. Male Sprague"Cawley* rats Comte! 10/6 ; 12/12 200p.p.m.. I day 12/12 ,, . 200D.DJC.. 3.daw . _ 'UK 200 p.pm.'tdfiys^.. 12/12 149 1.60 1.83 3.10 1.64 1.32 1.69 2.22 2.90 3.17 104 141 107 2.39 1.74 1.94 0.89 0.36 0.39 0J6 0.35 024 0.29 013 101 1.88 1.77 1.53 2.27 1.90 1.72 1.71 1.99 1.91 1.99 2.04 1.68 1.75 1.12 1.S4 8.31 8.24 9.32 15.69 7.42 . 7.71 13.40 13.63 4170 4474 4420 3500 4300 5340 4630 5870 1090 1210 1000 1270 1440 1830 1730 2030 817 543 779 1110 1330 1490 2010 2290 0.06 <0.01 <0.01 <0.01 0.14 0.47 0.13 0.12 0.0* 0.02 <0.01 <0.01 0.04 OOl 0.02 <0.01 <0.01 <0.01 <0.01 <0.01 X01 <0.01 <0.01 <0.01 0.12 0.03 0.10 0.16 0.07 0.01 0.02 <aoi 0.13 0.50 0.53 0.50 0.11 <0X2 0.21 0.22 0.94 2Ai 1.19 1.18 1.13 0.96 1.37 1.20 554 256 253 270 597 l 226231 289 461 509 439 595 670 990 830 880 156 249 245 256 906 572 694 773 ABUMh WgegtPOfl^felltatB10ealWe<n>itW| Vnrfwpri hftw I -- > Mlha/yflMWI day n4 IHTUH A wwyfM {?-riwy- coumirtn (MailjQiliii^'uKl an epemde hydrolase activity (with styrene 7,8-ode as substrate) were determined is the microsomal fractions. Glutathione transferase activity with 2,4-dinftro-chkxobenzene was measured in the cytosolic fractions. Values are means and S.D. of 3 individually measured animals or ac tivities of the pooled Cmcdoos from si surviving or at [east 10 animals. *Two different stocks of Sprague Dawley rats were used. The animals of experiment 8 were from a commercial source (WKJA, Sulzfeld. FRO) whereas those of experiment 9 wen bmd from obtained from Prof. C.Maltoni throughout the treatment in males at the high concentration of VDC, and was slightly increased in treated females. In rat kidney, monooxygenue activity was below the detec* don limit in control and in treated animals. Epoxide hydrolase activity was not measurably affected and giutathione transferase activity was slightly enhanced by the treat ment. In Table V, results of mutagenicity experiments using subcellular fractions from VDC-treated and-control animals are compared. A summary of these data is also included together with biochemical data in Table VI. With liver preparations as activating systems, the effects of VDC treatment were rather weak. This is in agreement with the weakness of the effects oa the activities of drug-metabolizing enzymes. Kidney prepara tions from control as wed as from VDC-treated rats and female mice did not activate VDC to a mutagen or had, at most, marginal effects. With male mice, kidney preparations from control animals showed a weak but definite activation 1035 AP00009954 F.Oock tt a/. Tabic V. Activation of VDC to a mutagen by liver and kidney postmito chondrial supernatant fractions from VDC-treated animals. Experiment number, animal* anti treatment Number of VDC-lndticed his4' revenue colonies Liver 25 mg 100 mg Kidney 29 mg 100 mg 1. Male Swiss Webster mice Control 490* 10 p.p.m.. 1 day 315* 10 p.p.m., 3 days 294* 10 p.p.m., 8 days 294* 4. Male Swiss Webster mice Control 544* 90 p.p.m., 1 day 456* 50 p.p.m., 3 days 300* 50 p.p.m., 8 days 446* 9. Male Swiss Webster mice Control 413* SO p.p.m., 1 day 328* 50 p.p.m., 3 days 370* 90 p,p,m., 8 days 302* 6. Female Swiss Webster mice Control 163* 50 p.p.m., 1 day 186* 90 p.p.m.', 3 days 198* 90 p.p.m., 8 days 236* 7. Female Swiss Webeter mice Control 398* 50p.p.m., 1 day 409* SO p.p.m., 3 days 306* 90 p.p.m., 8 days 362* 8. Male Sprague Dawley rats Control 50* 200 p.pjn., 1 day 73* 200 p.p.m., 3 day* 111* 200 p.p.m., 8 days 28* 9. Male Sprague Dawiey rats Control 200 p.p.m., 1 day 19* SO* 200 p.p-oi-, 3 days 61* 200 p.p.m., 8 days 17* 610* 518* 469* 508* 746* 565* 475* 556* 814* 408* 640* 982* 381* 453* 388* 545* 795* 633* 681* 751* 267* 83* 196* 190* no* 97* 16* 67* 89* 100* 39* 82* 62* 28 NS IONS 4 NS 28* 0 4 NS I NS 12 NS 12 NS -1 NS -2 NS -7 NS 16 NS 28 NS -2 NS 16 NS 30* 7 NS 8 NS 22* 2 NS 14* 7 NS 109* 102* 37* 108* 109* 60* 43* 87* 34* 12NS --7NS 31* 13NS -14NS -20NS 2NS -5NS -- SNS 18NS 20* --7NS 46* 24NS -- 11NS 42NS 1JNS 46* 33* Histidine-poor agar plate containing hii~ S. typhimurium TA100 and NADPH-fortified poatnatochoodrial fraction from 12.9 to 100 mg liver or kidney of control and VDC-tnated animals were exposed at 37C to 4900 p.p.m. VDC for 4 h. After a further incubation for 2 days at 37C, hb* revartant colonies were counted. Value* with liver preparedons are means from 4 parallel maihatipn* minus the corresponding number of mutants in the absence of VDC (95 -168). Values with kidney preparations are meant from 5 VDC-expoced plate minus means from 3 corresponding nan-etposed plates (112-197). Asterisks indicate statistically significant (p <0.09, t-ust) differences from non-otpoaed controls. NS, not significantly different from controls. Actmties ofdrugrnetabolizmgenzymes an shown in Table 111 osing the same numbering of (be experiments. of VDC to a mutagen. Preparations from VDC-treated animals, especially at the higher treatment concentration, ac tivated VDC less effectively than controls. Discussion In the present study, we have investigated VDC with respect to mutagenicity, effects on drug-metabolizing en zymes and the relationship between these two effects. Our results confirm earlier studies (17,19) which described rever sion of bacterial strains with the his G46 substitution muta tion; moreover, we found that VDC causes various other 1036 substitution and also frameshift mutations. Along with muta genicity studies in yeast (20) and in E. coli K.12 (32) these results indicate that VDC can induce a wide spectrum of dif ferent mutations. As was observed in previous studies, metabolic activation was required for mutagenicity to occur and preparations from mice showed stronger activation than preparations from rats and liver was more active than kidney, respectively. In contrast to previous studies, we investigated the same strains of animals which had been used for carcino genicity experiments. Interestingly, kidney preparations from male mice activated VDC, whereas preparations from female mice and from rats were practically inactive, which is in line with the results of the carcinogenicity studies. However, in spite of negative carcinogenicity results, Chinese hamster kidney preparations activated VDC even more than did preparations from male mice. Together with the relatively po tent activation of VDC by hepatic preparations, this indicates that forniation of mutagenic metabolites is not sufficient (but probably necessary) for carcinogenesis in a tissue to occur. In deed, in previous studies it hadbeen noticed that tumorigenic concentrations of VDC resulted in massive tissue damage (12), but caused only minimal DNA alkylation and DNA repair synthesis (33). Hence. non-genctic toxic effects may have been the decisive additional factor that tumours were formed only in the kidney in male mice, but not in other tissues and species which are able to activate VDC to a mutagen. An epoxide has been proposed as important intermediate in the mutagenicity of VDC (16,17^2^4). Urinary metabolites suggest the formation of glutathione conjugates (34--38), one of them probably derived from the epoxide. We therefore studied the effect of microsomal epoxide hydro lase and glutathione on the mutagenicity of VDC. Since addi tion of purified epoxide hydrolase from rat liver microsomes or an inhibitor of microsomal epoxide hydrolase, 1,1,1-tri- chloropropene 2,3-oxide (29), did not affect the mutagenicity of VDC, this enzyme does not seem to be important for the metabolic control of the mutagenic species derived from VDC. Glutathione reduced the mutagenicity of VDC as ex pected from available biochemical data (34--38) and from a mutation-decreasing effect by other thiols (17). We suspect that the stimulating effect which was observed in some ex periments with low concentrations of subceUular preparations in the presence ofglutathione was due to a stabilization of ac tivating enzymes, e.g. by inhibition of lipid peroxidation. To date there are no reports about the effects of VDC on drug-metaboliaiig enzymes. Such effects may be important in chronic exposures, e.g. in carpinogenidty experiments, because they could potentiate or protect against toxic effects. Various increases and decreases in enzyme activities occurred after treatment of animals with VDC (Table VI). In the mouse, effects were found in the kidney whilst the enzyme ac tivities remained virtually unchanged in the liver. In kidney of male mice, decreases in all three enzyme activities were observed after short exposure. An indication of an increase was seen only with epoxide hydrolase after prolonged treat ment. In contrast, in female mice, only the monooxygenase activity was decreased and the activities of the other two en zymes were increased (with the exception of a short in termediate decrease in epoxide hydrolase activity in one ex periment). We suggest that (0 the decreases in activity were caused by cytotoxic effects and, therefore, were predominant In the males, which are more susceptible than females, and (ii) AP00009955 r VtayUdeoe dtlwMc hqim induction and imii^nifc Uj Table VI. Summary Of (he effects observed in animals after treatment with VDC. Animals, treatment Mortality (in 8-day treatment group) Male Swiss Webster mice, 10 p.p.m. 0<R# Male Swiss Webster mice, 30 p.p.m. 69 Female Swiss Webster mice, SO p.p.m. 0% Male Sprague Dawfcy rats, 200 p.p.m. 90% Chsngs lit activiciH Liver Monooxy- Epoxide genase hydrolase . s 1 Glutathione Activation transferase of VDC Kidney Monooxy genase Epoxide hydrolase (-) (I) (I) (t) 1* 1* GhitatMone Activation transferase of VDC 0) 1 0 0 I, Increase; l, decrease: =. no change; 0. not measurable or extremely weak activities. Brackets Indicate that the effect was weak in comparison with the variation and that, therefore, the interpretation must be considered as tentative. Asterisks signify that the indicated changes were dearly predominant, bat that the effects were complex and also included opposite changes at some stages of the time oourse. The results are shown In detail in the Tables JV and V. the mechanisni(s) which increases enzyme activities, possibly enzyme induction, is not related to toxicity, but directly to the VDC concentration; furthermore, the two mechanisms may counteract each other; thus, toxidty may inhibit enzyme in* ducdon while increases in activities such as glutathione transferase may protect against toxicity. Hence, differences in susceptibility in the uninduced state may be potentiated by in creases in the activities of protecting enzymes in the kss susceptible animals. The rat tolerates higher concentrations of VDC than mice (of both sexes) and was therefore treated with 200 p.pun. VDC compared with 50 p.p.m. for mice. Enzyme activities in the rat were affected in the kidney and also in the liver. A moderate decrease in hepatic monooxygenase activity was observed after the first treatment and, later, a gradual in crease of hepatic epoxide hydrolase and glutathione transferase activity was seen. In the kidney, an increase in glutathione transferase, but not in epoxide hydrolase activity occurred; monooxygenase activity was not measurable either in control or in treated animals. Since 7-ethoxycoumarin is a broad spectrum substrate which is dealkylated by various cytochromes P-450, it is likely that enzymes which activate VDC also metabolize 7-ethoxycoumarm. Thus, low mono oxygenase activity may be one contributing factor for the low susceptibility of rats, compared to mice, to VDC. In conclusion, in this comparative biochemical study we have made various observations which can be associated with the susceptibility of tissues to the carcinogenic action of VDC. The results indicate relatively complex relationships zySW and carttflBfeehiClly. Altogether, flUl dam S>UpP8rt the notion that tag effects are necessary for the formation of tumours by vS& m-NWttahAn rtf mutagens and (ii) tissue damage. 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