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REVIEW ARTICLES VINYL CHLORIDE--PART 1: METABOLISM The period of phrenetic activity--or at least sustained debate--on the status of vinyl chloride (VC) seems to have passed. Since techno* logical feasibility eventually formed the basis of the first generation of VC legislation, the authorities are now biding their time, pending the next generation of biological data, which will allow an assessment of the hazards associated with the currently permitted levels. We last reviewed VC early in 1976 (BIBRA Bull. 1976, 15, 6 & 72). In the inter vening period the biochemists and microbiologists have demonstrated particular interest in this monomer. Dose-related metabolism A preliminary study by Dow chemical USA on the fate of unlabelled VC in the rat indicated that its metabolism was dependent on the level of exposure (Hefner et al. Ann. N.Y. Acad. Sci. 1975, 246, 135). In the range 50-105 ppm the half-life was 86 min, whereas in the range 2201167 ppm the half-life was 261 min. Following up this work, the same group investigated the metabolism of *4C-labelled VC 3n the rat (Watanabe et al. Toxic, appl. Pharmac. 1976, 37, 49). Rats exposed to 10 ppm VC for 6 hr eliminated 68% of the absorbed radioactivity in their urine and 2% (as VC) in the expired air over 72 hr; 6-hr exposures to 1000 ppm resulted in 56% of the dose appearing in the urine with 12% in the expired air as VC. The pulmonary excretion of the VC followed firstorder kinetics with similar half-lives at both treatment levels; 20*4 min at 10 ppm and 22*4 min at 1000 ppm. Elimination of l4C in the urine was more complex, being described by a two-exponential equation; but the rates of the initial phase were comparable! at 10 ppm the half-life was 4-6 hr while at 1000 ppm it was 4*1 hr. After 72 hr, 14-15% of the. administered radioactivity of both the high and low doses was recovered from the carcases, indicating, when correction is made for the amount of VC metabolized, that there is increased storage of radioactivity at higher doses. The metabolism of VC administered by other routes appears also to be dose-dependent. Indeed an even greater disparity between the metabolism of high and low doses seems to follow oral administration. In another study by Watanabe et al. (ibid 1976, 36, 339) from 59 to 68% of an oral dose of 0-05 or 1 mg VC/kg was converted to non-yolatile urinary metabo lites, with 9-13% appearing as expired CO, and only 1-2% being excreted by the lungs as unchanged VC. By contrast, 11% of the highest dose of 100 mg/kg was excreted in the urine, and 67% was excreted as VC (and 3% as C02) in the expired air. The pulmonary excretion at 100 mg/kg was biphasic, with half-lives of 14*4 and 40*8 min, whereas at 0*05 and 1 mg/kg it was monophasic with a half-life of about 55 min. / ucc 033851 ' 7~1*' M/f 8 r > Similar findings were reported by the ICI workers. Green & Hathway (Chemlco-BioL. Interactions 1975, 11, 545), low intragastrlc doses of VC being eliminated principally in the urine whilst higher doses were mainly excreted unchanged via the lungs. About three-quarters of an intubated dose of 250 (ig [ *4C]VC/kg was converted to non-volatile urinary metabo lites and 12-15% was excreted in the expired air as CO, together with a few per cent as unchanged monomer. By contrast, at a dose of 450 mg/kg pulmonary excretion predominated, with 90% of the radioactivity being associated with unchanged VC and less than 1% with C02. The pulmonary elimination of monomer, shown to be proportional to the log of reciprocal dose, was completed within 5 hr, whereas both the excretion of C0Z via the lungs and the elimination of non-volatile metabolites in the urine continued over 72 hr. Similar excretion kinetics were observed in rats that, had been pretreated for 60 days with unlabelled VC at doses up to 300 mg/kg/day (Green & Hathway, loc.cit.). Withey (J. Toxicol, envir. Hlth 1976, 1, 381) was unconvinced that' intubation was an acceptable method of administering VC in a metabolic investigation, for he observed wide inter-animal variability in hisstudies in which rats with a cannulated jugular vein were intubated with aqueous and oil solutions of VC. The uptake of VC was rapid, peak concentrations being achieved within 10 min, with blood levels varying from 6 to over 40 Jig VC/ml. - The metabolism of VC given by iv and ip injection was also studied at ICI (Green & Hathway, loc.cit.). Almost all of a small (250 pg/kg) iv dose was found in the expired air as VC. About 43% of the same dose given by ip injection was excreted in the expired air., as VC, another 43% being converted to urinary metabolites and 11% to CO . An ip dose of 450 mg VC/kg was almost totally excreted (96%) as VC In the expired air, with only 2*6% being metabolized to non-volatile species excreted in the urine. Identity of metabolites ' ' Identification of the urinary metabolites was attempted in both the Dow and ICI studies.' In the oral and inhalation studies of Watanabe et al. (loc.cit.), most of the radioactivity excreted in the urine of rats was associated with three compounds, the proportions of which were unaffected by the dose or by the route by which the VC was administered.-' W-Acetyl-S-(2-hydroxyethyl)cysteine accounted for 29-40% and thiodiglycollic acid for 18-26% of the radioactivity detected in the urine, with the unidentified third compound being present at levels of from 30 to 39%. In the oral experiments, the three metabolites represented from 91 to . 95% of the total radioactivity excreted in the urine, whilst in the inhalation experiments the corresponding figure was 96-97%. The first report by Green a Hathway (loc.cit.) of their investi gations of the metabolism of orally administered [14C]VC (150 mg/kg) in the rat, noted that the three major urinary metabolites were thiodiglycollic acid (47% of the urinary radioactivity), S- (2-chloroethyl)cysteine (23%) and W-acetyl-S-(2-chloroethyl)cysteine (23%). The remaining radio activity in the urine was associated with urea (6%), glutamic acid (0*5%), chloroacetic acid (0*5%), methionine (trace) and serine (trace). Watanabe et al. (Toxic, appl. Pharmac. 1976, 36, 339) were critical of these results, suggesting that the chloroethyl compounds were artefacts formed UCC * % 033652 I by the reaction of the hydroxy analogues with the hydrochloric acid gas used in the derivatization procedure. This criticism was accepted by Green & Hathway who reported in their most recent paper (Chemico-Biol. Interactions 1977, 17, 137) that the major urinary metabolites of VC in the rat were AT-acetyl-S-(2-hydroxyethyl)cysteine, the related tf-acatylS-vinylcysteine and thiodiglycollic acid'. Further elucidation of VC's metabolic pathway(s) was attempted by Green & Hathway (1977, loc.cit.), who measured the yields of thiodigly collic acid in the urine of rats given the various possible metabolites intragastrically at high doses. Thiodiglycollic acid in 0*5% theoretical yield was isolated in the urine of rats dosed with S-(2-hydroxyethyl) cysteine, a result said to be highly significant in view of the cysteine derivative's instability in even mild reaction conditions. Furthermore, as high yields of the acid were found in those animals dosed separately with chloroacetaldehyde, chloroacetic acid and 5-(carboxymethyl) cysteine, it was suggested that all these compounds might lie on a common meta bolic pathway linking VC with thiodiglycollic acid. Nevertheless, the fact that only small amounts of chloroacetic acid (<0*1%) were detected in the body fluids of animals treated with the high doses of VC, backed by evidence from the metabolism of chloroacetaldehyde (and vinylidene chloride), suggested that chloroacetic acid is not a major VC metabolite. Green & Hathway (1977, loc.cit.), believed that in rats, chloroethylene oxide was formed from VC and might then be transformed spontaneously into chloroacetaldehyde. The chloroacetaldehyde or chloroethylene oxide reacts principally with glutathione, catalysed by glutathione-S-epoxide transferase, to form 5-(2-acetal)cysteine with subsequent formation of S-(2-hydroxyethyl)cysteine and its JT-acetyl derivative, S-(carboxymethyl) - cysteine and thiodiglycollic acid. ~ It was the view of Hefner and his colleagues (loc.cit.) in 1975 that VC was metabolized at low exposures, below 100 ppm, to 2-chloroethanol, chloroacetaldehyde and monochloroacetic acid via the alcohol dehydrogenase pathway. Only small amounts of the chloroacetic acid were formed at these low doses, due to the rapid reaction of chloroacetalde hyde with the sulphydryl groups of glutathione and cysteine. It was thought that at higher exposures'>(200-1000 ppm) direct epoxidation by microsomal oxidases occurred, with rearrangement of the resulting chloro ethylene oxide to chloroacetaldehyde. However, the more recent study of Bolt et al. (Arch. Tox. 1976, 35, 153) showed that in rats exposed to the monomer at an initial atmospheric concentration of 50 ppm in a closed system, the uptake of VC was completely blocked initially by 3-broraophenyl4(5)-imidazole or 6-nitro-l,2,3-benzothiadiazole, both inhibitors of cytochrome P-450-dependent metabolism and was increased by DDT pre treatment, suggesting that the microsomal oxidases may be operative even at low doses. Weaker inhibitors of this enzyme system, such as SKF-525A or 5,6-dimethyl-l,2,3-benzothiadiazole, produced a measurable but lower order of inhibition. In the original Dow work (Hefner et al. loc.cit.), . SKF-525A had no effect on the metabolism of low doses of VC (65 ppm) but did slightly decrease metabolism at VC exposures of the order of 1000 ppm. Cytochrome P-450 involvement Liver microsomal fractions have been shown to interact with VC in vitro (Salmon, Cancer Lett. 1976, 2, 109). The addition of VC to rat-liver microsomes and NADPH produced a Type 1 spectral shift, similar ucc 033653 10 to that seen for phenobarbital, thus indicating direct involvement of a cytochrome P-450 species. Other in vitro studies have also demonstrated that the oxidation of VC to non-volatile products was dependent both on microsomal enzymes and on NADPH (Kappus at al. Toxic, appl. Pharmac. 1976/ 37, 461). It has been suggested that VC metabolites can destroy cytochrome P450. Ivanetich et al. (Biochem. biophys. Res. Commun. 1977, 74/ 1411)/ for example/ showed not only that the binding and metabolism of VC in vitro was fully inhibited by SKF-525A, indicating the involvement of the cyto chrome P-450 system, but that the VC metabolites reduced the level of both cytochrome P-450 (reduced to P-420) and microsomal haem. The levels of cytochrome b5 or NADPH-cytochrorae c reductase were unaffected by exposure to the monomer. Decreases in the level of cytochrome P-450 and in the total activity of hepatic microsomal enzymes were observed in rats 24 hr after they had been exposed for 6 hr to a 5% VC atmosphere (Reynolds et al. Res. Commun. chem. Path. Pharmac. 1975, 12, 685). The selective enzymatic deactivation was consistent with a cytochrome P-450-centred activation of VC to reactive electrophiles. The destruction of cytochrome P-450 was found to be inhibited by glutathione in the study of Ivanetich et al. (loc.cit.), whereas Guengerich & Strickland (Molec. Pharmacol. 1977, 13, 993) reported that neither added reduced glutathione nor cysteine offered any protection against the VC-mediated destruction. The loss of cytochrome P-450 activity was attributed by Guengerich & Strickland (loc.cit.) to the destruction of haem, and not to lipid peroxidation or the binding of electrophiles to free sulphydryl groups. Since the destruction of P-450 required all the components necessary for mixed-function oxidation, it was said that the oxidative metabolism of VC by cytochrome P-450 was necessary for the observed destruction. The investigators did not consider, however, that the two commonly proposed VC-metabolites, the epoxide and chloroacetaldehyde, were the agents res ponsible for the cytochrome P-450 loss, as neither compound proved par ticularly effective at destroying either free or P-450-bound haem in vitro. Consequently it was postulated that different mechanisms underlie VC's mutagenic activity and its ability to destroy P-450. The latter is localized at the activating enzyme, consistent with a highly reactive species, whereas mutagenesis seems to require a metabolite stable enough to be transported from its site of activation to interact with nucleic acids or associated macfomolecules. Comment The view that the microsomal mixed-function oxidases play an active and possibly the major role in the metabplism of VC monomer has received support from the demonstration that a microsomal-oxidase pathway operates at low VC exposures. The results of in vitro studies have also indicated that VC is metabolized by enzymes of the liver microsomes, in particular by a cytochrome P-450 species, to give initially chloroethylene oxide and 2-chloroacetaldehyde. Further investigation into VC-mediated des truction of cytochrome P-450 is required, but it would seem that here, too, a cytochrome enzyme is Involved in the formation of the active meta bolite (s) . The metabolic studies conducted by Dow and ici have confirmed the dose-dependence of the fate of VC in the rat. It would seem, on the evidence now available, that in the rat there is effectively a single saturable metabolic pathway for VC. As the dose of VC.increases, an 1JCC 033654 M "n increased proportion escapes metabolism and is eliminated unchanged in the expired air. The fact that the major urinary metabolites--probably derived from the conjugation of chloroethylene oxide or 2-chloroacetaldehyde with glutathione--appear to be unaffected both qualitatively and quantitatively by dose tends to oppose the hypothesis of alternative pathways varying in importance in relation to dose. If there are a number of important metabolic pathways* then they appear to be leading to the same end products* an unlikely,' although not impossible* occur rence. [j. Hopkins] GETTING RID OF PENTACHLOROPHENOL As a bactericide* fungicide and herbicide, pentachlorophenol (pcp) finds use in a wide range of applications! in particular* it has become an important wood preservative. Its potential significance as a major environmental contaminant must therefore be recognized. PCP appears to be readily absorbed through the skin and has been held responsible for aplastic anaemia, muscular paralysis and sensory loss in factory workers who failed to use adequate protective clothing and who worked in poorly ventilated areas (BIBRA Bull. 1965* 4, 314). The toxicity of PCP to rats was shown to be greater when the compound was inhaled than after oral or subcutaneous administration (ibid 1977, 16, 204) . The question of PCP toxicity is complicated* however* by the fact that technical-grade PCP is frequently contaminated with varying amounts of toxic by-products* including other chlorinated phenols and* in some cases, chlorodibenzo-p-dioxins and chlorodibenzofurans. Thus Kimbrough & Linder (Toxic, appl. Pharmac.. 1978, 46, 151) found that whereas technical PCP fed for 8 months at a level of 500 or 100 ppm in the diet caused pronounced morphological changes in the livers of rats and some change was noted even with 20 ppm* no change was detec ted in rats fed 20 or 100 ppm purified PCP and only slight changes were apparent in those given 500 ppm in the diet. Nevertheless* PCP itself has been shown to be an extremely effi cient uncoupler of oxidative phosphorylation in mitochondria (Weinbach* J. biol. Chera. 1954* 210, 545 Weinbach a Garbus, Nature* Lond. 1969* 221, 1016) and to be capable of disturbing microsomal detoxication mechanisms in the liver (Arrhenius et al. Chemico-Biol. Interactions 1977, 18, 35). By gas-chromatographic analysis of the sub-cellular fractions of the livers of rats treated orally with 0*15 mmol (c. 40 mg) PCP/kg* the latter group demonstrated that* compared with the cytosol, the mitochondria contained markedly lower concentrations of PCP and the microsomes showed a high accumulation {Idem, ibid 1977, 18, 23), suggesting that the effects of PCP on the detoxication, functions of the endoplasmic reticulum are at least as relevant physiologically as the compound's capacity for mitochondrial uncoupling.. The fate of a single oral dose (10 or 100 mg/kg) of ^C-labelled pure PCP was studied in rats for 9 days, with a final examination of tissues for residual activity (Braun et al. Toxic, appl. Pharmac. 1977, 41, 395). The primary route of excretion of PCP was through the kid neys , with most of the remaining fraction of the dose appearing in the ucc 033655 evidence of direct mutagenic activity, its activity was doubled by the addition of the S-9 fraction from a PCB-treated rat. The results of a similar study by Andrews et al. (Nutation Res. 1976, 40, 273) seem to be anomalous, in that vc at levels of up to 15% in air were shown to be directly mutagenic in TA1535 and an S-9 fraction from PCB-treated rats produced only a slight enhancement. However, the differences between these two sets of results may partly be explained by the different in cubation times involved. Vinyl chloride is not thought to be mutagenic per so. The increase in the number of histidine revertants seen when VC is incubated with strains TA1530, TA1535 or TA100 in the absence of any mammalian metabo lizing system is thought to be a result of the non-enzymic breakdown products of VC or of compounds formed by the bacterial enzyme system (Bartsch et al. loc.cit.). Aqueous solutions of VC (initial concentration 0*083 M) gave no evidence' of mutagenicity when incubated with S. typhimurium strains TA1530, TA1535 or G46 together with a fortified 9000-g liver supernatant from phenobarbital-treated mice (Bartsch et al. loc.cit.). A similar lack of activity of VC in solution (at an initial concentration of 0*022 M) was reported by Elmore et al. (Biochim. biophys. Acta 1976, 442, 405) in TA100. It was thought that the inactivity of VC in these systems may have been caused by rapiddiffusion of monomer from the liquid phase into the atmosphere. *= 5. typhimurium strains TA1530, TA1535, TA100 and G46 respond with varying sensitivity to monofunctional alkylating .agents (base-pair substitutions or deletions). Strains TA1536, TA1537 and TA1538, which are specifically reverted by frameshift mutagens, were unaffected by 20% VC atmospheres even in the presence of liver fractions from rats or mice (Bartsch et al. loc.cit.; Rannug et al. loc.cit.). The effects of the various liver fractions seen in the studies reviewed by Bartsch & Montesano (Mutation Res. 1975, 32, 93) indicate that the mixed-function oxidases are responsible for the conversion of VC to mutagenic metabolites in mammals. Garro and his colleagues (ibid 1976, 38, 81), however, did not believe that the hepatic fractions were exerting their effect by an enzymatic mechanism. In contrast to the results of Bartsch et al. (loc.cit.) who reported that 20% VC in air induced a mutagenic response in TA1530 only when an NADPH-generating system was present, Garro et al. (loc.cit.), testing 75% VC in air in the same Salmonella strain, found that the activating effect of the S-9 microsomal fraction on VC mutagenesis was independent of the NADPHgenerating system (NADPH being a necessary co-factor for the mixedfunction oxidases) and was largely unaffected by a thermal treatment that would be sufficient to inactivate all enzyme activity. The in creases in VC-induced mutagenicity observed when the liver fractions were obtained from either untreated or PCB-treated animals were similar, although the PCB would be expected to increase the mixed function oxi dase content of the liver. Garro et al. (loc.cit.) considered, there fore, that the liver fraction might have been increasing the mutageni city of VC non-enzymatically, perhaps by promoting the formation of free radicals. A free-radical generating system, riboflavin irradiated with UV light, doubled the number of VC-induced revertants in th 5. typhimurium ucc 033656 of the carbonic anhydrase activity in the testis was contributed by the erythrocytes in the circulating blood and how much was derived from the local tissues. From the findings, it was concluded that' a1terations in the carbonic anhydrase activity of the testes following Cd administration reflected changes in the blood content and therefore in erythrocyte car* bonic anhydrase in the organ. The exact role played by these and possibly other factors in the damaging effects exerted on the testis by Cd remains to be established. The problem continues to attract wide interest, however, and it may be that the placing of a few more key pieces will enable the rest of the jigsaw to be fitted into place. x [p. Cooper] VINYL CHLORIDE--PART 2: MUTAGENICITY Last month, we reviewed the more important metabolic studies on vinyl chloride (VC) . Now we turn to mutagenicity. This aspect of VC's biological profile has been extensively investigated, particularly in bacteria. Mutagenicity in Salmonella typhimurium The preliminary studies of mutagenicity involved the Ames test. When VC, at a concentration of 204 (v/v) in air, was incubated with Salmonella typhimurium strain TA1535 and a mammalian metabolizing system, namely a fortified post-mitochondrial rat-liver supernatant, the number of histidine revertants was increased to three times the spontaneous mutation rate (Rannug et al. AMBXO 1974, 3, 194). An increase over the spontaneous mutation rate also occurred in strains TA1535, TA1530 and G46 when they were exposed to a similar VC atmosphere in the presence of a 9000-g mouse-liver supernatant (S-9 fraction) fortified with an NADPHgenerating system (Bartsch et al. Int. J. Cancer 1975, 15, 429). Tha greatest response was seen with TA1530, VC treatment (for 48 hr) being associated in this case with a 28-fold increase in the number of histidine revertants. The VC concentration of the incubation medium below the 204 atmosphere was found to be 0-004 H at equilibrium. Although exposure to 20% VC in air proved to be mutagenic towards TA1530 in the absence of metabolic activation, mutagenic activity was doubled by the addition of liver fractions from phenobarbital-treated mice. The highest mutagenic responses occurred in the presence of a 9000-g supernatant or with the recombined microsomal and soluble protein fraction in the presence of an NADPH-generating system. Purified microsomal fractions were less effi cient activators, and soluble liver proteins (100,000-g supernatant) alone were almost without effect. The addition of alcohol dehydrogenase and NAD+ to either the fortified post-mitochondrial or the 100,000-g liver supernatant did not increase the mutation rate (Bartsch et al. loc.cit.). In the TA1535 and TA100 strains, McCann et al. (Proc. natn. Acad. Sci. U.S.A. 1975, 72, 3190) found that although a VC atmosphere (20% v/v) gave UCC 033657 inactive in this system (Elmore et si. loc.cit.). Chi roethylene xide, at concentrations of up to 25 pM, and 2-chloroacetaldehyde, at concen trations up to 12*8 yM, also produced a dose-dependent induction of 8azaguanine- or ouabain-resistant mutants in Chinese hamster V79-4 cells in vitro, but 2-chloroethanol or chloroacetic acid were inactive at concentrations nearly 100 times higher (Huberman et al. Int. J. Cancer 1975, 16, 639). In the presence of mouse-liver microsomes, vinyl chloride atmospheres (up to 50% v/v) significantly increased the forward mutation frequency of Schizosaccharamyces pombe and induced gene conversion in Sacchazomyces cerevisiaa (Loprieno et al. Mutation Res. 1976, 40, 85). No mutagenic; activity was apparent in the absence of the microsomal fraction. chloroethylene oxide was directly and highly mutagenic in these test systems (idem. Cancer Res. 1977, 36, 253). In either the presence or absence of microsomes, a commercial aqueous solution of 2-chloroacetaldehyde exhibited only feeble genetic activity, whilst 2-chloroethanol was totally inactive. Since a concentration of 0*05 mM chloroethylene oxide induced the same mutagenic frequency as 50 mM VC, it seems likely that all the activity of the monomer in this system could be ascribed to the oxide. In a host-mediated assay in the mouse, VC given at 700 mg/kg by gavage significantly increased the mutation frequency of Sch. pombe, which had been injected into the peritoneal cavity during 12 hr of treat ment (Loprieno et al. 1976, loc.cit.). Methyl methanesulphonate, used as a positive control, proved to be almost 200 times as active as VC. No significant increases in forward mutation were seen 3 or 6 hr after an ip injection of 250 mg 2-chloroacetaldehyde/kg (Loprieno et al. 1977, loc. cit.). Either in ethanolic solution or in the gaseous state, VC induced no detectable mutagenic change in two strains of Nevrospora crassa, a eukaryotic micro-organism (Drozdowicz & Huang, Mutation Res. 1977, 48, 43). The addition of am S-9 liver fraction from either control or phenobarbitone-treated rats had no effect. The apparent lack of sensitivity of this __ system may have been due to the inability of VC and its metabolites to penetrate the conidia. A significant increase in the frequency of recessive lethals over that in controls was observed in mala Drosophila melanogaster exposed to VC at 850 ppm for 2 days and there was a slight increase in mutation rate on exposure to 30 ppa for 17 days (Verburgt a Vogel, ibid 1977, 48, 327). No further enhancement in the recessive lethal incidence was seen after 2-day exposures to VC levels in excess of 10,000 ppm. It was suggested that above this concentration the Drosophila enzymes were no longer capable of metabolizing (activating) additional VC. In contrast to the recessive lethal' assay, negative results were obtained in tests on domi nant lethals, translocations and entire and partial sex-chromosome loss in Drosophila exposed to VC at 30,000 ppm for 2 days (Verburgt & Vogel, loc.cit.). VC has failed to produce dominant lethals when tested in a mammalian system. Male CD-I mice, exposed to 3000, 10,000 or 30,000 ppm VC for 6 hr/day for 5 days, were housed for 5 days in each of the following 8 wk ucc 033653 (TA1530) system, and this stimulation was further enhanced by the presence of a photopolymerization accelerator--JV,JV,tf',Ar'-tetramethylethylenediaraine. Mutagenicity of VC metabolites The two suspected VC metabolites, chloroethylene oxide and 2-chloroacetaldehyde each directly increased the number of histidine revertants in strain TA1530 of S. typhimurium. At initial concentrations of 0*4 pmol/ml the oxide was a^out twice as active as the aldehyde with respect to mutagenicity, but chloroethylene oxide exhibited a lower toxicity; 11% of a cell population survived an oxide concentration of 0*4 pmol/ml while less than 0-004% of the Salmonella cells exposed to the same concentration of the aldehyde survived (Malaveille at al. Blochera. biophys. Res. Commun. 1975, 63, 363). Chloroethylene oxide was strongly mutagenic towards TA1535. At an initial concentration of 0-45 mM, the spontaneous mutation rate of thre mutants/10 cells was increased to 96/10 surviving cells. .Chloroacetaldehyde at an initial concentration of 0-5 mM only increased the spontan eous mutation rate approximately three-fold (Rannug et al. Chemico-Biol. Interactions 1976, 12, 251). It was calculated that chloroethylene oxide, with a half-life in solution of only a few minutes at 37C, was some 450 times as mutagenic as the aldehyde on a mol:mol basis. The oxide (tested at 0-26 M) and 2-chloroacetaldehyde (0-1 M) were also shown to be muta genic in Salmonella strain TA100 (Elmore et al. loc.cit.). Because of its instability, an increased number of histidine revertants were observed when the oxide was pre-incubated with the test micro-organism at 3C. All of the four possible forms of 2-chloroacetaldehyde exhibited a mutagenic response against TA100; the pure compound was more?active than the mono mer hydrate which in turn was more active than the dimer hydrate and trimer, probably demonstrating the importance of the intact electrophilic carboxyl group in eliciting a mutagenic response (Elmore et al. loc.cit.). In the presence of a post-mitochondrial mouse-liver fraction, 2chloroethanol (108 pmol/plate) increased the number of. histidine re vertants in the TA1530 strain of S. typhimurium about ten times; distinct mutagenic activity, but of a lower- order, was also observed in the absence of metabolic activation (Bartsch et al. loc.cit.). Chloroacetic acid induced neither a direct nor a tissue-mediated response in this strain. In the study by McCann et al. (loc.cit.) 2-chloroethanol was weakly muta genic against TA100, inducing 0-6 revertant colonies/pmol, and demonstrated a trace of activity in TA1535. Microsomes significantly increased the number of compound-induced histidine revertants in TA100 whilst horseliver alcohol dehydrogenase together with NADH had no analogous effect. Elmore et al. (loc.cit.) observed no mutagenic response when 2-chloro ethanol was incubated at 1 mM with strain TA100. When tested by Rannug et al. (1976, loc.cit.) 2-chloroethanol and chloroacetic acid were without effect on TA1535 at concentrations up to 1*5 mM; much higher concentrations of 2-chloroethanol (1 M) proved to be weakly mutagenic. Mutagenicity in other systems In Bacillus subtilis, chloroethylene oxide and 2-chloroacetaldehyde selectively inhibited strain MC-1, a mutant lacking recombination repair of DNA; chloroethanol, chloroacetic acid and VC itself (in solution) were ucc 033659 with successive pairs f untreated females (Anderson et ai. Mutation Res. 1976* 40, 359). Using the female mice as the indicator organism there was no evidence that VC had any mutagenic effects on any maturation stage of spermatogenesis. There was no significant increase in the number of post-implantation early foetal deaths* no evidence of pre-implantation egg loss (except at the highest VC dose* where the loss showed a low level of significance but could largely be attributed to a single female) * nor any reduction in fertility. A dominant lethal effect was clearly demonstrated in the positive controls given a single ip injection of 200 mg cyclophosphamide/kg or five oral doses of 200 mg ethyl methanesulphonate/kg. In most of the Salmonella studies* VC demonstrated activity only in the presence of a microsomal liver fraction. The Ames test has given conflicting evidence on metabolism; whilst the studies of Bartsch efc al. (Xoc.cit.), in line with the other in vitro studies* suggested that the microsomal fraction of the liver was acting enzymatically* the experiments of. Garro et al. (loc.cie.) indicated, so far uniquely* that a freeradical mechanism was responsible for the conversion of VC to its active metabolites. VC elicited a mutagenic response in a simple eukaryotic system* demonstrating activity in Sch. pombe and Saccharomyces cerevisiae both in vitro in the presence of liver microsornes and in a host-mediated assay* The incidence of recessive lethal mutation was increased in Drosophila treated with 30 ppm VC for 17 days. Chloroethylene oxide and 2-chloroacetaldehyde have proved to be mutagens--the oxide particularly so--in prokaryotic micro-organisms* namely the strains of Salmonella susceptible to frameshift mutagens and B. subtilis. This evidence further implicates chloroethylene oxide as the active VC .metabolite. No .evidence of activity was seen in the dominant lethal assay in mice. However* further investigations of mutagenicity in mammals are re quired before an assessment of the genetic hazards presented by VC to man will be possible. [j. Hopkins] O ucc 033^0 REVIEW ARTICLES VINYL CHLORIDE--PART 31 MACROMOLECULAR BINDING Chloroethylene oxide and chloroacetaldehyde are probably the principal metabolites of vinyl chloride (BIBRA Bull. 1979, 18, 7). Evidence of the binding of these metabolites to cellular macromolecules/ particularly the alkylation of the nucleic acids, may explain the mutagenicity of vinyl chloride (Ibid 1979, 18,-79).' In vivo studies The irreversible binding of VC metabolites to nucleic acid and protein has been demonstrated in vivo (Bolt, et al. in IARC Scientific __ Publn no.13; IARC, Lyon, 1976, p.151). In rats exposed to atmospheres containing about 140 ppm [14c]vC, roughly half of the radioactivity found in the liver microsomes immediately after the 5-hr exposure was irreversibly bound to protein, although the major proportion of labelled material in the liver cells was present in the cytosol. Immediately after exposure to 44 ppm [ 14C]VC, from 10 to A0% of the total activity in the tissues was irreversibly bound, while at 48 hr the proportion of bound activity had increased to 70%, although the amount of irreversibly bound VC metabolites stayed the same. Radioactivity was also found in the liver nucleic acids. The peak incorporation of activity into DNA (0-23% total liver activity) occurred immediately after exposure, whereas the specific labelling of the RNA (0-8% of initial activity of the liver) was at a maximum after 24 hr and decreased more slowly. Bolt e al. (loc.cit.) calculated that as no more than 1% of the labelled VC would enter the one-carbon pool to be incorporated into protein and nucleic acid, this mechanism could not explain the total high level of binding observed in the study. It was possible that the one-carbon pool could account for the amounts of label found in the nucleic acid of the liver. However, the authors thought this unlikely since the speed by which this activity decreased was faster than normal DMA turnover and the rate of incorporation of label was very different in RNA and DNA derived from the same cellular nucleotide pool. In further studies at the same institute (Lalb & Bolt, Arch. Tox. 1978, 39, 235), liver RNA isolated from rats exposed to VC atmospheres was shown to contain labelled l,N^-ethenoadenosine and 3,N4-ethenocytidine, the alkylation products of adenine and cytosine by chloroethylene oxide and chloroacetaldehyde, as well as the physiological bases. The time- \ course of dealkylation was found to differ in the two bases. Ninety-two hours after exposure, the level of labelled ethenoadenosine was only one- fifth of its original value; ethenocytidine proved more resistant to repair and the levels of radioactivity associated with this component remained constant over the same period--possibly an indication of the relative importance of cytidine alkylation. /ffrX It* ucc " 033661 fit J Watanabe et al. (Toxic, appl. Pharraac. 1978, 44, 571) exposed Sprague-Dawley rats to 1-5000 ppm l4C-labelled VC for 6 hr. The amount of radioactivity bound to macromolecules in the liver did not increase in proportion to the increase in exposure concentrations.. Covalent binding to hepatic macromolecules was related to the amount of VC metabolized. There was no detectable binding of *4C to either DNA or RNA in the liver, in contrast to the results of Bolt et al. (loc.cit.). The authors thought this difference in results might be explained by the higher specific activity used by Bolt et al. They also offered the suggestion that the incorporation observed in the earlier work might have originated from the one-carbon pool, but this would not explain the alkylated bases isolated by Laib & Bolt (loc.cit.). Watanabe et al. (loc.cit.) concluded that covalent binding to hepatic nucleic acids occurred to only a limited degree under their experimental conditions but they pointed out that this did not exclude the possibility of other interactions which might result in the loss of the ability to control replication. Hepatic glutathione content was depressed at exposure concentrations above 100 ppm, and according to these authors reactive metabolites of VC may be detoxified by glutathione, suggesting that the carcinogenicity of VC may be related to a decreased ability to detoxify reactive metabolites. The in vivo binding of VC metabolites to DNA has also been investi gated in mice (Osterman-Golkar et al. Biochem. biophys- Res. Commun. 1977, 76, 259). BALB mice took up radioactivity about twice as fast as the CBA and ATL strains. Levels of S-(2-hydroxyethyl)cysteine, N1and w3-hydroxyethylhistidine or ff7-hyaroxyethylguanine were measured in the hydrolysates of proteins and nucleic acids taken from mice exposed to 98-302 ppm; these compounds were formed from the chemical (sodium borohydride) reduction of 2-oxoethyl groups introduced into the cell macromolecules as a result of the VC treatment. The histidine and guanine derivatives and small amounts of hydroxyethylcysteine were detected in hydrolysates of haemoglobin and liver DNA. The authors calculated that the absolute and relative amounts of alkylated products supported the hypothesis that the main reactive metabolite of VC is chloroethylene oxide. In the BALB mice, the two hydroxyethylhistidines could be detected in small amounts in the protein from the testes. In vitro studies The binding of the metabolites of VC to cellular macromolecules has also been studied by incubating rat-liver microsomes with NADPH, polyadenosine and [1,2- **c3vc (Laib & Bolt, Toxicology 1977, 8, 185). The radioactivity identified in the enzymic hydrolysates of the poly adenosine was irreversibly attached to 1,W6-ethenoadenosine. An analogous reaction occurred when polycytidylic acid was incubated with VC and the microsomal system, labelled 3, N4 -ethenocytidine moieties being formed (Laib & Bolt 1978, loc.cit.). The reaction of chloroacetaldehyde with calf-thymus DNA at pH 4*5 gave a modified DNA product (Green & Hathway, Chemico-Biol. Interactions 1978, 22, 211). Enzyme hydrolysis of the DNA produced a mixture of naturally occurring deoxyribonucleosides and chloroacetaldehyde reaction products, predominantly ethenodeoxycytidine and thenodeoxyedenosine. ucc 033662 V ** V ** 0 , 188 The same products resulted from hydrolysis of liver DNA prepared from rats that had been exposed to 250 ppm VC in drinking-water for 2 yr. According to the studies of Kappus et al. (Toxic, appl. Pharmac. 1976, 37, 461), the alkylation of protein and uptake of VC by rat-liver microsomes is dependent on concentration, incubation time, enzymatic activity, NADPH and oxygen, and is almost completely blocked by CO. These authors found that only about 1% of the VC taken up by the micro somes became irreversibly bound, a finding that contrasts with the corresponding figure of around 50% observed in the in vivo studies of Bolt et al. (loc.cit.). Glutathione added to the microsomal incubation mixture decreased the level of irreversible protein binding. When trichloropropene oxide, an inhibitor of epoxide hydrase, was present, the irreversible protein binding increased two-fold, even though VC uptake by the microsomes was unaffected. . Other in vitro studies have produced evidence of chloroethylene oxide's participation in the metabolism of VC. The reaction product of chloroacetaldehyde or chloroethylene oxide with adenosine was tentatively" characterized by Barbin et al. (Biochem. biophys. Res. Commun. 1975, 67, . 596) as 3,B-ribofuranosylimidazo-(2,1-i)-purine (l,W6-ethenoadenosine) ; a product with the same Rp value and elution characteristics on a Sephadex column was formed when VC was incubated with adenosine in the presenc of a microsomal fraction. Barbin et al. (loc.cit.J passed a mixture of VC and air or oxygen into a medium containing both-"liver microsomes from a phenobarbitone-treated mouse and an NADPH-generating system. The volatile metabolite trapped by reaction with 4 -(4-nitrdbenzyl) pyridine (4-NBP) in ethylene glycol had a UV absorption spectrum identical to the product formed from the reaction of chloroethylene oxide (but not 2-chloro acetaldehyde) with 4-NBP. The evidence from these studies strongly supports the view that chloroethylene oxide is the major reactive VC metabolite. While it is clear that metabolites of VC do bind to cellular macromolecules in rat and mouse liver, the extent of nucleic acid binding has not been clearly resolved. [J. Hopkins] BLACK MARKS FOR MBK The industrial solvent methyl n-butyl ketone (MBK) has been implicated in cases of peripheral neuropathy among Spray-painters and other workers (BIBRA Bull. 1976, 15, 360) and has been shown to induce axonal degeneration of the central nervous system and polyneuropathy in experimental animals (ibid 1977, 16, 147) . The metabolic fate and disposition of MBK in the rat has been studied by DiVincenzo et al. (Toxic, appl. Pharmac. 1977, 41, 547) in male rats dosed by gavage with [1-***C]MBK at 20 r 200 mg/kg. Absorption UCC 033663