Alan S. Wright

The Relationships between Alkylation of Haemoglobin and DNA in Fischer 344 Rats Exposed to [14C]Ethylene Oxide

The quantification of products (adducts) of chemical reactions between geno-toxic chemicals and Hb may provide a basis for determining individual exposures to such agents and similarly the determination of Hb dose (in vivo) might be used to estimate the doses of ultimate genotoxic agents delivered to DNA (Ehrenberg et al 1974). The validation of this approach for its applicability to man relies on the study of the relationships in experimental species. Published data relating Hb- and tissue DNA- doses of EO are limited (Osterman-Golkar 1983; Segerback 1983). Thus the current study, designed to investigate the general utility of Hb as a DNA monitor in cases of exposure to EO, aimed to relate exposure doses of EO to the doses delivered in vivo to Hb and to DNA in F344 rats. The relationships obtained in this study can be compared with those obtained in other species with a view to determining the variation. These results are based on measurements of DNA doses of EO in brain, lung, liver, spleen, kidney and testis and Hb doses in blood of male F344 rats exposed to inhaled [14C]EO. The inhalation concentrations of EO used in this dosimetry study (1, 10 and 33 ppm, v/v) were similar to the lower range of exposures (10, 33 and 100 ppm, v/v) in the Bushy Run carcinogenicity study (Snellings et al 1984) and were selected with a view to investigating possible saturation kinetics at low doses.

The chemical and biochemical reactivity of dichlorvos

The chemical structure, reactivity and metabolic fate of the insecticide dichlorvos (2,2-dichlorovinyl dimethyl phosphate) are discussed in relation to the possible genotoxicity of this and other methyl phosphate triesters. Recent attempts to demonstrate the methylation of DNA following exposure of bacteria and animals to dichlorvos are reviewed. On the basis of comparative data relating mutagenesis to methylation reactions, it seems entirely appropriate to conclude that the mutagenicity of dichlorvos to bacteria is due solely to methylation of the bacterial DNA under the conditions of these tests. However, the methylation of mammalian DNA could not be demonstrated under realistic exposure conditions (when the alkylating mutagen methyl methanesulphonate afforded clearly measurable methylation). The failure to detect methylation by dichlorvos in vivo is attributed to the operation of highly efficient enzyme-catalysed biotransformations which rely largely on the phosphorylating reactivity of dichlorvos. The biotransformation pathways, characterised mostly in the rat, appear to be common also to pig, mouse, hamster, and man.

Molecular dosimetry of DNA adducts in rainbow trout (Oncorhynchus mykiss) exposed to benzo(a)pyrene by different routes

Farm raised rainbow trout (Oncorhynchus mykiss) were exposed by various routes to benzo(a)pyrene (BP) as a representative carcinogenic polycyclic aromatic hydrocarbon (PAH). Following exposure of fish to the chemical by intraperitoneal (i.p.) injection, 32P-postlabelling studies indicated that non-feral trout were relatively resistant to the formation of BP-DNA adducts in liver. No adducts were detected in fish exposed to single doses (20 mg/kg) of BP. Multiple exposures (e.g. 2×25 mg/kg) were necessary in order for adducts to be detected, indicating that induction of the metabolising enzymes required for the bioactivation of BP is necessary. These studies provided reference information on DNA adducts for comparison with data from subsequent experiments at environmentally realistic low level exposures. Two types of low level aquatic exposure were carried out. The first procedure exposed fish for 30 days to a nominally constant low level (1.2 and 0.4 μg/l) of a homogeneous dispersion of BP in water, to simulate low level aquatic environmental exposures. Following 32P-postlabelling analysis of the liver DNA of exposed fish, BP-DNA adducts were not detected. In the second procedure, fish were exposed to a constant low level of BP (ca. 0.5 μg/l) for 15 days then to a pulse (60 μg/l) which was allowed to naturally decline (to ca. 2 μg/l) during a further 15 days. Following this exposure, significant levels of BP-DNA adducts were detected in livers of trout. The effect of dietary exposures was investigated by feeding trout a diet containing either 58 μg or 288 μg BP per day for 6 days, equivalent to total doses of 43 mg/kg and 216 mg/kg. In both cases BP-DNA adducts were detected in livers of exposed fish. The results provide useful information on the types of exposures to PAHs which may pose a genotoxic risk to fish in the environment.

In vivo alkylation studies with dichlorvos at practical use concentrations

Twenty male CFE rats were exposed to atmospheres containing 0.064 microgram/l of [Me-14C] dichlorvos (113 Ci/mol) for 12 h. Analysis of the DNA and RNA from the total soft tissues of these rats revealed no methylation of the N7 atom of guanine moieties. The limits of detection of methylation were one methyl group per 6.0 X 10(11) and per 2 X 10(9) nucleotide units for DNA and RNA, respectively. Only 0.000001% of the administered dose would have needed to react with DNA in order to produce detectable methylation of this macromolecule. The exposure period employed in this study (12 h) constituted a significant fraction of the half-life of 7-methylguanine moieties in DNA (3 days). On the basis of this information and the extremely rapid metabolism of dichlorvos in a wide range of mammalian tissues and species it was concluded that dichlorvos does not methylate the nucleic acids of mammalian tissues when it is inhaled continuously at practical use concentrations.

The protective action of glutathione on the microbial mutagenicity of the Z- and E-isomers of 1,3-dichloropropene

The Z(cis)- and E(trans)-isomers of 1,3-dichloropropene (DCP), in confirmation of previous reports, caused dose-dependent increases in the numbers of reverse mutations in Salmonella typhimurium TA100 in the presence and absence of a 9000 X g supernatant fraction (S9) from the livers of Aroclor-treated rats. The relevance of these findings to mammals is uncertain, not least because of major differences in the metabolism of the DCPs in the microbial assay systems and in vivo. For example, (Z)-DCP is efficiently detoxified in mammals by the operation of a glutathione (GSH)-dependent S-alkyl transferase. It is possible that such detoxification could proceed only very slowly in the microbial assays because the concentrations of GSH could be severely rate-limiting even in those assays fortified by the addition of S9. The results obtained in the current study demonstrate a dramatic reduction in the microbial mutagenicity of both (Z)- and (E)-DCP when the concentration of GSH in the microbial assays was adjusted to a normal physiological concentration (5 mM). However, this protective action of GSH was at least as effective in the absence of S9 as in its presence, suggesting that it was not mediated by mammalian GSH transferase. There appears to be little or no GSH alkyl or aryl transferase in the cytosol of S. typhimurium TA100, but intracellular GSH is present at a concentration similar to that found in mammalian cells. Since the uncatalysed reaction between the DCPs and glutathione is relatively slow, the effect is not due simply to their destruction by GSH. It is possible that a physiological concentration of extracellular GSH maintains the intracellular GSH in a reduced form in which its nucleophilic thiol group competes effectively with the nucleophilic centres in the bacterial DNA for the haloalkenes. The current results highlight the efficiency of GSH-linked systems in affording protection against the genotoxic action of the DCPs. It may be presumed that their operation would exert a major limiting effect on the genotoxicity of (Z)- and (E)-DCP in mammals.

Microbial mutagenicity studies with (Z)-1,3-dichloropropene

This study has confirmed that the direct mutagenicity previously observed when S. typhimurium TA100 was treated with (Z)-1,3-dichloropropene (DCP) was in fact due to trace impurities. These impurities result from autoxidation of (Z)-1,3-DCP and have now been identified. Both (Z)- and (E)-2-chloro-3-(chloromethyl)oxiranes (DCP oxides) were identified as significant products during this autoxidation. The mutagenic impurities formed by autoxidation were completely removed by adsorption chromatography on silicic acid. (Z)-1,3-DCP purified in this way had no direct-acting mutagenicity towards S. typhimurium TA100. However, (Z)-1,3-DCP undergoes mono-oxygenase-catalysed conversion into bacterial mutagens in the presence of S9 fraction or washed microsomes from rat liver. The glutathione-linked conjugation systems of mammalian tissues provided efficient protection against this indirect mutagenic action. However, the low concentration of glutathione in standard bacterial mutagenicity assays limits the glutathione S-alkyl transferase-catalysed detoxification of (Z)-1,3-DCP and its primary bioactivation product(s). When the concentration of glutathione was adjusted to the normal physiological concentration, the mono-oxygenase-dependent mutagenic action of (Z)-1,3-DCP was virtually eliminated. These results therefore are consistent with the view that bacterial mutation assays are only qualitative indicators of potential mammalian genotoxicity.

The effect of hepatic microsomal monooxygenase induction on the metabolism and toxicity of the organophosphorus insecticide chlorfenvinphos.

The induction of rat liver microsomal monooxygenase by pretreatment of rats with dieldrin affords a 10-fold protection against the acute toxic effects of the organophosphorus insecticide, chlorfenvinphos. Metabolism studies were carried out to confirm that the protection was due to an enhanced rate of detoxification (via oxidative deethylation). At low doses of chlorfenvinphos (2.5 mg . kg-1), dieldrin pretreatment caused minimal changes in the metabolic profiles. However, at a higher dose (13.2 mg . kg-1), giving clinical signs of intoxication in the control animals, the dieldrin pretreated rats produced 5 times more deethylchlorofenvinphos than did the control animals. The results support the conclusion that the effect of enzyme induction on the metabolism of substrates of that enzyme are dose-dependent. Alterations in metabolism, therefore, are not an automatic consequence of enzyme induction.

Studies on the dermal and systemic bioavailability of polycyclic aromatic compounds in high viscosity oil products

Abstract The assessment of skin penetration by viscous oil products is an important element in the risk assessment of these materials where skin contact is likely. Systemic bioavailability (body uptake) is viewed as a good indicator of skin penetration following cutaneous exposures. The results of this study provide quantitative information on the influence of viscosity on the bioavailability of a specific polycyclic aromatic compound (benzo(a)pyrene) in base oils, residual aromatic extracts and bitumens following skin exposures to mice. The materials studied were a base mineral oil (viscosity 32 cSt at 35 °C), a 1:1 blend of the mineral base oil and a residual aromatic extract (198 cSt), several residual aromatic extracts (ca. 5000 cSt, 35 °C) and a range of bitumens (0.65–69 × 106 cSt, 35 °C). These were each spiked with 0.1% radiolabelled benzo(a)pyrene, as a representative carcinogenic polycyclic aromatic compound, then used for cutaneous exposures to mice. The results indicate that as viscosity increased in the range ca. 30 to 5000 cSt (base oil to residual aromatic extract) the uptake of the radiolabelled benzo(a)pyrene into blood was reduced by ca. fivefold. Further increases in viscosity from ca. 5000 to 69 × 106 cSt (i.e. residual aromatic extract to bitumen) resulted in a further but smaller (ca. twofold) reduction in uptake. The relationship between the amounts of free benzo(a)pyrene measured in blood and viscosity showed the same trend. This trend was also mirrored by the degree of binding of benzo(a)pyrene metabolites to DNA in skin. The findings in mouse skin in vivo indicate that viscosity can significantly affect skin penetration and systemic bioavailability of polycyclic aromatic compound components of oil products. Results obtained with viable human skin in vitro also showed that the bioavailability of benzo(a)pyrene was reduced by the viscosity of the oil product matrix. It is thus necessary to take account of physical properties such as viscosity in the overall risk assessment of viscous oil products, particularly in the case of very viscous materials such as bitumens. The significantly reduced bioavailability of hazardous compounds from undiluted materials is thus an important factor to consider when assessing the risks from dermal exposures.

Genotoxicity studies with mineral oils; effects of oils on the microbial mutagenicity of precursor mutagens and genotoxic metabolites.

In vitro genotoxicity assays are extensively used to predict carcinogenic activity in vivo. The standard microbial mutagenicity assays however often fail to yield positive results with mineral oils which are carcinogenic to mice in long-term skin-cancer studies. A comprehensive programme of studies has therefore investigated the basis of this apparently anomalous behaviour. This investigation has addressed the possible effects of oils on the bioactivation of precursor mutagens and the disposition of mutagenic metabolites by studying the microbial mutagenicity of selected precursor mutagens (benzo[a]pyrene, benzo[a]anthracene, 2-aminoanthracene and 2-naphthylamine) and intrinsically reactive mutagens [+/- )-benzo[a]pyrene-4,5-oxide and (+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene) in the presence and absence of mineral oils. Notably the mutagenicity associated with the deliberate additions of these mutagens or precursor mutagens to oils was readily detected by the microbial assays. The mutagenicity of only one of the precursor mutagens, benzo[a]pyrene, was significantly reduced by the oils, and then only in the standard plate-incorporation assay. Interestingly the degree of suppression appeared to be related to the polycyclic aromatic hydrocarbon content of the oils. In the case of 2-aminoanthracene large enhancements in its mutagenicity were observed in the presence of oils. These latter findings appear to be due to effects of oils on the bioactivation of precursor mutagens rather than on the disposition of their bioactivation products. The mutagenicity of intrinsically reactive mutagens, of a type generated by bioactivation of polycyclic aromatic hydrocarbons, was not significantly reduced in the presence of mineral oils. This indicates that it is unlikely that components in oils trap or facilitate the deactivation of ultimate mutagens whether these pre-exist in the oil or are formed from precursors by bioactivation in the in vitro test system. Viewed overall these results suggest that mineral oils judged to be carcinogenic on the basis of in vivo studies in mouse skin may possess only very weak genotoxic potential. While this potential is likely to be a prerequisite for carcinogenic action, the current results cause attention to be focussed on other factors, e.g. promotion, as potentially important determinants of the carcinogenic potencies of mineral oils in mouse skin.

Use of organ cultures in human risk assessment: Comparisons of benzo[a]pyrene-DNA adducts in mouse and human skin.

Human and mouse skin explant systems have been developed as models that qualitatively mimic the genotoxic metabolism in vivo of benzo[a]pyrene, a representative carcinogenic polycyclic aromatic hydrocarbon. High-performance liquid chromatography profiles of DNA adducts isolated from CD-1 mouse skin treated with [(3)H]benzo[a]pyrene either in vivo or in vitro were qualitatively very similar. (32)P-Post-radiolabelling and thin-layer chromatography analysis of these DNA adducts also yielded profiles that were qualitatively similar both in vivo and in vitro. The presence of a component that did not appear to correspond to those detected by direct labelling was also revealed by (32)P-postlabelling. These approaches have been applied in order to develop valid risk models for chemical carcinogens, and in addition, to provide valuable reference standards for the detection of human genotoxic metabolism. Thus the coupling of human skin explant systems with (32)P-postlabelling techniques has provided a direct in vitro model for the prospective detection of human carcinogens.