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 [3H]benzo[a]pyrene either in vivo or in vitro were qualitatively very similar. 32P-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 32P-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 32P-postlabelling techniques has provided a direct in vitro model for the prospective detection of human carcinogens.

Advances in our understanding of the mechanisms of chemical carcinogenesis are now being applied to improve the quality of prospective risk assessment. The contribution of Ehrenberg and his colleagues (at the University of Stockholm) probably represents the most comprehensive application of mechanistic knowledge to this field during the past 20 years. The strategic approach developed by the Swedish group was based on the identification of differences between man and experimental risk models in factors that determine the relationships between exposure and biological response and the development of methods to compensate for these differences. Many of the critical stages in chemical carcinogenesis and the cellular determinants of these stages have now been identified. As a first step in seeking to improve risk assessment, Ehrenberg introduced the target dose concept, in which the doses of carcinogens penetrating to the cellular target (DNA) are determined. This approach provides an improved basis for determining exposures to carcinogenic agents and also for compensating for species differences in factors such as metabolism that determine the relationships between exposure dose and the dose at the critical target. The target dose concept is now widely accepted and has led to the development of new biomedical monitoring techniques, based, for example, on the measurement of haemoglobin adducts, which are now being applied to detect and identify genotoxic hazards. The introduction of the target dose concept has led to significant improvements in the quality of prospective risk assessment. Further improvements necessitate procedures to compensate for differences between man and prospective risk models in factors that determine subsequent stages of the carcinogenic process. Ehrenberg has proposed that the rad-equivalence approach may be of value in this respect. Its application has accurately predicted the incidence of leukaemias in occupational cohorts which had exposures to ethylene oxide in common. The possible general applicability of this approach is discussed.

In order to obtain information on the genotoxic metabolism of carcinogens in human skin in vivo, model in-vitro systems have been developed to mimic in-vivo metabolism qualitatively. Direct labelling (3H and 14C) and 32P-postlabelling analyses of benzo[alpha]pyrene (BP)-DNA adducts in human skin explants, CD1 mouse explants and CD1 mouse skin in vivo have thus allowed comparisons of the genotoxic metabolism of BP in mouse and human skin.

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.

Human exposures to high concentrations of vinyl chloride (VC) have been associated with the occurrence of angiosarcoma of the liver (Maltoni et al. 1982; Creech and Johnson 1974; Spirtas and Kaminski 1978). A programme of studies in this laboratory is investigating the relationships between exposure to VC, the dose at critical cellular targets, e.g. DNA, and the carcinogenic response in experimental species, e.g. rat. A key aspect of this work has been the development of highly sensitive quantitative methods for measuring the DNA dose of vinyl chloride in target tissues.

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.

A systematic molecular phenotyping approach based on two-dimensional gel electrophoresis is being applied in an attempt to identify protein changes associated with malignant transformation. Using the C3H10T1/2 mouse cell line, two-dimensional polypeptide maps of the non-transformed cell line, several chemically transformed lines and a tumour cell line were compared. Although there is a large degree of similarity between the protein profiles of all cell lines, clear differences are evident. Initial results are consistent with the view that many of the protein changes are incidental to malignant transformation. Changes induced by 3-methylcholanthrene are retained after transplantation of the cells into nude mice.

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.

Methods for the detection of in vivo alkylation of nucleic acids are discussed. Alkylation of mammalian DNA at N-7 of guanine provides the most sensitive and least equivocal evidence of methylation by an exogenous chemical or its metabolite. No 7-methylguanine was detected in the combined DNA from the lungs, livers, hearts, brains, kidneys, testes and spleens from 20 rats exposed to 0.064 micrograms l-1 of [Me-14C]-dichlorvos at a specific radioactivity of 113 Ci mol-1 for 12 hrs.

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 dosedependent. Alterations in metabolism, therefore, are not an automatic consequence of enzyme induction.

We report in this work an environmentally benign zinc mediated synthesis of aryl and benzyl phosphorochalcogenoates in ethanol within a short reaction time. In vitro antimicrobial study along with statistical analysis and seed germination assay were performed. These chalcogenophosphates possess strong antimicrobial activity against the reference strains. The antibacterial activity was determined against four standard strains (Bacilus subtilis, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa). The antifungal activity was evaluated against one fungal strain Candida albicans.

This paper reports on the biochemical aspects of a wide-ranging study of the effects of long-term (ca. 6 years) exposure of male rhesus monkeys to technical grade dieldrin at dietary concentrations from 0.01 to 5 ppm. Apart from small changes associated with the hepatocellular endoplasmic reticulum, the overall study revealed no effects that could be attributed to dieldrin. Increases in the activity of the liver microsomal monooxygenase system, determined in vitro with a range of substrates, provided the most sensitive criteria for an effect. Significant increases, to a maximum of threefold, were observed in the 1.75- and 5-ppm treatment groups. These increases in monooxygenase activity were paralleled by increases in cytochrome P-450. There was no effect on glucose 6-phosphatase, a constitutive enzyme of the hepatocellular endoplasmic reticulum, or on the activities of hepatic alkaline phosphatase and succinic dehydrogenase. Although the increases in microsomal enzyme activity were associated with small increases in microsomal protein, there was no significant effect on liver weight nor on the DNA content of liver. The concentrations of dieldrin in the tissues of the 0.1-ppm treatment group of monkeys were very similar to those measured in humans receiving a similar daily intake of dieldrin per kilogram body weight. The concentrations of dieldrin in these monkey livers were, however, ca. 200 times higher than in male rats receiving a daily intake of dieldrin ca. 3 times higher than the monkeys and were very similar to the concentrations measured in the livers of male mice ingesting ca. 50 times more dieldrin per kilogram body weight per day than the monkeys. The dietary intake of dieldrin required for the induction of the rhesus monkey liver microsomal monooxygenase system was 25 to 30 μg/kg body w/day, approximately 300 times greater than that of the general human population in 1966–1967. The corresponding threshold concentration in the liver was 6 to 7 ppm. Such concentrations are associated with marked microsomal enzyme induction and overt liver enlargement in rats and mice. The results obtained with rhesus monkeys and the absence of any detectable changes in the livers of humans exposed to high endogenous concentrations of dieldrin point to a slow rate of metabolic clearance of dieldrin in these primate species and to a low sensitivity of their livers to this compound.

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.

In 1987, the US Environmental Protection Agency (EPA) classified aldrin and dieldrin as category B2 carcinogens, i.e. probable human carcinogens, based largely on the increase in liver tumors in mice fed either organochlorine insecticide. At that date, the relevant epidemiology was deemed inadequate to influence the cancer risk assessment. More time has now elapsed since early exposures of manufacturing workers to aldrin/dieldrin; therefore, updated epidemiological data possess more power to detect exposure-related differences in cancer risk and mortality. Also, recent experimental studies provide a plausible mode of action to explain the mouse specificity of dieldrin-induced hepatocarcinogenesis and call into question the relevance of this activity to human cancer risk. This monograph places this new information within the historic and current perspectives of human cancer risk assessment, including EPA’s 1996 Proposed Guidelines for Carcinogen Risk Assessment. Updated epidemiological studies of manufacturing workers in which lifetime exposures to aldrin/dieldrin have been quantified do not indicate increased mortality or cancer risk. In fact, at the middle range of exposures, there is evidence of a decrease in both mortality from all causes and cancer. Recent experimental studies indicate that dieldrin-induced hepatocarcinogenesis in mice occurs through a nongenotoxic mode of action, in which the slow oxidative metabolism of dieldrin is accompanied by an increased production of reactive oxygen species, depletion of hepatic antioxidant defenses (particularly α-tocopherol), and peroxidation of liver lipids. Dieldrin-induced oxidative stress or its sequelae apparently result in modulation of gene expression that favors expansion of initiated mouse, but not rat, liver cells; thus, dieldrin acts as a nongenotoxic promoter/accelerator of background liver tumorigenesis in the mouse. Within the framework of EPA’s Proposed Guidelines for Carcinogen Risk Assessment, it is proposed that the most appropriate cancer risk descriptor for aldrin/dieldrin, relating to the mouse liver tumor response, is ‘not likely a human carcinogen’, a descriptor consistent with the example of phenobarbital cited by EPA.

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 ingestion of dieldrin by male rats, male mice, female beagles and male rhesus monkeys resulted in proliferation of the smooth endoplasmic reticulum of liver parenchymal cells. This change was associated with an enhanced activity of the liver microsomal mixed-function oxidase system. The dieldrin-induced alterations in liver subcellular structure and function were reversible in the rat, mouse and dog, although regression was slow in the latter species. Regression was not studied in monkeys. Phenobarbitone elicited a similar response to dieldrin in the rat, mouse and dog. Compound comparisons were not attempted in monkeys. The effects of the carcinogen, 4-amino-2,3-dimethylazobenzene, on mouse liver contrasted with the effects of dieldrin or phenobarbitone. Exposure to the carcinogen resulted in a depression of liver glucose-6-phosphatase activity with no increase in the activity of the liver microsomal mixed-function oxidase system. The significance of the results is discussed.