Metabolism of the bay-region diol-epoxide of chrysene to a triol-epoxide and the enzyme-catalysed conjugation of these epoxides with glutathione.

Metabolic activation of chrysene in mouse skin appears to involve r-1,t-2-dihydroxy-t-3,4-oxy-1,2,3,4-tetrahydrochrysene (anti-chrysene-1,2-diol 3,4-oxide) and 9-hydroxy-r-1,t-2-dihydroxy-t-3,4-oxy-1,2,3,4-tetrahydrochrysene (anti-9-OH-chrysene-1,2-diol 3,4-oxide). The enzyme-catalysed conjugation of these epoxides with [35S]glutathione has been studied in experiments in which the glutathione conjugates were separated by h.p.l.c. and examined by fluorescence spectrophotometry. Both anti-chrysene-1,2-diol 3,4-oxide and anti-9-OH-chrysene-1,2-diol 3,4-oxide formed conjugates nonenzymically and both were shown to be substrates for rat liver glutathione transferases. When anti-chrysene-1,2-diol 3,4-oxide was incubated with [35S]glutathione and a rat liver microsomal metabolizing system, glutathione conjugates with h.p.l.c. and fluorescence spectral characteristics identical to those of conjugates formed from both anti-chrysene-1,2-diol 3,4-oxide and anti-9-OH-chrysene-1,2-diol 3,4-oxide were detected. This finding provides evidence that anti-chrysene-1,2-diol 3,4-oxide can be further metabolized to the triol-epoxide, anti-9-OH-chrysene-1,2-diol 3,4-oxide by rat liver microsomal systems.

Mutagenic and cell-transforming activities of triol-epoxides as compared to other chrysene metabolites.

The syn- and anti-isomers of the bay-region diol-epoxides of chrysene and of 3-hydroxychrysene and their metabolic precursors have been investigated for mutagenicity in Salmonella typhimurium (reversion to histidine prototrophy) and V79 Chinese hamster cells (acquirement of resistance to 6-thioguanine) and for transforming activity in M2 mouse prostate cells. Other known and potential chrysene metabolites have been included in mutagenicity experiments. Direct mutagenic activity in S. typhimurium TA 100 exhibited, in order of potency, anti-triol-epoxide greater than syn-triol-epoxide greater than anti-diol-epoxide greater than syn-diol-epoxide greater than chrysene 5,6-oxide much greater than chrysene-1,2-quinone, chrysene-3,4-quinone, and chrysene 5,6-quinone. Chrysene, the six isomeric chrysenols, and the trans-dihydrodiols [trans-1,2-dihydroxy-1,2-dihydrochrysene (chrysene-1,2-diol), trans-3,4-dihydroxy-3,4-dihydrochrysene, trans-5,6-dihydroxy-5,6-dihydrochrysene, and 9-hydroxy-trans-1,2-dihydroxy-1,2-dihydrochrysene (9-hydroxychrysene-1,2-diol)] were inactive per se but were activated to mutagens in the presence of reduced nicotinamide adenine dinucleotide phosphate-fortified postmitochondrial fraction (S9 mix) of liver homogenate from Arochlor 1254-treated rats. Chrysene, 3-hydroxychrysene, chrysene-1,2-diol, and 9-hydroxychrysene-1,2-diol were activated efficiently; the other compounds were activated weakly. In S. typhimurium TA 98, the mutagenic activities of the chrysene derivatives were weak in comparison with those in the strain TA 100. trans-3,4-Dihydroxy-3,4-dihydrochrysene (in the presence of S9 mix) was the most efficacious mutagen in strain TA 98. The relative mutagenic potencies of the directly active compounds differed from the results obtained in strain TA 100, in that in strain TA 98 the anti-diol-epoxide was more mutagenic than the triol-epoxides and chrysene 5,6-oxide was more mutagenic than syn-diol-epoxide and syn-triol-epoxide. In V79 cells, the order of mutagenic potency was: anti-triol-epoxide greater than anti-diol-epoxide greater than syn-triol-epoxide greater than syn-diol-epoxide greater than chyrsene 5,6-oxide greater than chrysene-1,2-diol (in the presence of S9 mix) greater than 9-hydroxychrysene-1,2-diol (in the presence of S9 mix) greater trans-3,4-dihydroxy-3,4-dihydrochrysene in the presence of S9 mix). Chrysene, 3-hydroxychrysene, 5-hydroxychrysene, and 6-hydroxychrysene showed no mutagenic effects in V79 cells, either in the presence or absence of S9 mix.(ABSTRACT TRUNCATED AT 400 WORDS)

Comparative studies of the metabolic activation of chrysene in rodent and human skin.

Metabolism and activation of chrysene was examined in mouse, rat and human skin using a short-term organ culture technique. Mouse skin released larger quantities of free dihydrodiols into the culture medium than either rat or human skin and greater quantities of chrysene metabolites became covalently bound to the DNA of mouse skin. The stereochemistry of the chrysene-1,2-diol that was formed by each skin type was examined using high-performance liquid chromatography (HPLC) with a chiral stationary phase to resolve the enantiomers. It was found that in each case the (-)-enantiomer predominated. When hydrolysates of DNA extracted from rodent or human skin that had been treated with 3H-labelled chrysene were chromatographed on Sephadex LH-20 columns, the elution profiles of the hydrocarbon-DNA adducts were found to vary between the species studied. Further examination using HPLC showed that some of the adducts formed in skin had the chromatographic characteristics of adducts formed when the anti-isomer of the 'bay-region' diol-epoxide of chrysene (r-1,t-2-dihydroxy-t-3,4-oxy-1,2,3,4-tetrahydrochrysene) reacted with DNA and that others had the characteristics of triol-epoxide adducts.

The formation of 9-hydroxychrysene-1,2-diol as an intermediate in the metabolic activation of chrysene.

9-Hydroxy-trans-1,2-dihydro-1,2-dihydroxychrysene (9-hydroxychrysene-1,2-diol), which may be the triol involved in the formation of a chrysene triol-epoxide-DNA adduct in mouse skin, was not detected when chrysene was incubated with rat-liver microsomal preparations. In separate experiments an excess of synthetic 9-hydroxychrysene-1,2-diol was added during the incubation of 3H-labelled chrysene with rat-liver microsomes and was then re-isolated. The triol was found to contain a radioactive product that had chromatographic properties identical to those of 9-hydroxychrysene-1,2-diol when examined by reverse-phase h.p.l.c., both before and after acetylation, by normal-phase h.p.l.c. and by t.l.c. both before and after oxidation. When treated with m-chloroperoxybenzoic acid, the synthetic 9-hydroxychrysene-1,2-diol formed products that possessed alkylating activity and that reacted with DNA in vitro. Examination of the triol-epoxides produced by oxidation of a mixture of synthetic and metabolic 9-hydroxychrysene-1,2-diol by t.l.c. suggested that the anti-isomer was formed.

Microsome-mediated reactions of phenols of polycyclic hydrocarbons with DNA.

Phenolic metabolites of 7 polycyclic hydrocarbons were incubated with rat-liver microsomal fractions in the presence of DNA. Hydrocarbon-nucleoside adducts with chromatographic properties similar to those of diol-epoxide-deoxyribonucleoside adducts, were detected in hydrolysates of DNA that had been incubated with phenols of benzo[a]pyrene, benz[a]anthracene and chrysene. Adducts were not detected when phenols of phenanthrene, pyrene, dibenz[a,c]anthracene or dibenz[a,h]anthracene were further metabolised. The possible contribution of phenolic metabolites to the carcinogenic activity and DNA binding of polycyclic hydrocarbons is discussed.

Metabolic activation of chrysene in mouse skin: evidence for the involvement of a triol-epoxide.

All three possible dihydrodiols of chrysene and a chrysene triol, formed from the further metabolism of the chrysene-1,2-diol, were detected when ether extracts of mouse skin that had been treated with 3H-labelled chrysene were examined by h.p.l.c. The major deoxyribonucleoside-hydrocarbon adducts present in hydrolysates of DNA isolated from the mouse skin were examined by chromatography on Sephadex LH20 and by h.p.l.c. on Zorbax ODS. One adduct had chromatographic properties identical to those of the major adduct formed when r-1,t-2-dihydroxy-t-3,4-oxy-1,2,3,4-tetrahydrochrysene reacts with DNA. A second major adduct was present that had chromatographic properties that were indistinguishable from those of an adduct that was formed when either chrysene-1,2-diol or 3-hydroxychrysene were incubated with DNA in a rat liver microsomal metabolising system. The results provide evidence that this new adduct is formed via the reaction of a 'triol-epoxide', that appears to be 9-hydroxy-chrysene-1,2-diol 3,4-oxide, with DNA in mouse skin.

Metabolic activation of chrysene by hamster embryo cells: evidence for the formation of a 'bay-region' diol-epoxide-N2-guanine adduct in RNA.

Ribonucleoside-hydrocarbon adducts present in hydrolysates of RNA isolated from hamster embryo cells treated with 3H-labelled chrysene were examined by chromatography on Sephadex LH20 and by h.p.l.c. on Zorbax ODS. Two adducts formed in cells had chromatographic properties identical to those of two synthetic adducts formed when r-1,t-2-dihydroxy-t-3,4-oxy-1,2,3,4-tetrahydrochrysene (antichrysene 1,2-diol 3,4-oxide) reacted with poly G in vitro. Another adduct formed in cells had chromatographic properties identical to those of a synthetic adduct formed when antichrysene 1,2-diol 3,4-oxide reacted with poly A. In addition to the characterized adducts, other minor adducts were detected whose structures are not known. The structure of the more abundant guanosine--hydrocarbon adduct formed in cells was investigated by determining its pK values and stability in 1 M KOH. The structures of the synthetic guanosine--hydrocarbon adducts were investigated by 1H-n.m.r. spectroscopy. The data show that, in the hydrocarbon--guanosine adducts studied, the hydrocarbon moiety is attached to the exocyclic amino group of guanine.

Mechanism of esophageal tumor induction in rats by N-nitrosomethylbenzylamine and its ring-methylated analog N-nitrosomethyl(4-methylbenzyl)amine.

The metabolism of the esophageal carcinogen N-nitrosomethylbenzylamine (MBN) and its ring-methylated analog N-nitrosomethyl(4-methylbenzyl)amine (4-MeMBN) was investigated in male Wistar rats. When given in the drinking water, both compounds have been shown to induce a high incidence of esophageal carcinomas but, after systemic administration of equimolar doses, 4-MeMBN is considerably less toxic and carcinogenic than is MBN. Following a single i.v. injection, 4-MeMBN disappeared from serum faster than did MBN. After 5 hr, neither compound was detectable in serum. Within 12 hr after a single i.v. injection (0.017 mmol/kg) of [methyl-14C]-MBN, 49% of the radioactivity was exhaled as 14CO2, and less than 5% was in the urine, compared with only 13% as 14CO2 and 65% in the urine after an equimolar dose of 4-Me[methyl-14C]MBN. The urinary metabolite of 4-MeMBN was identified as its benzoic acid derivative. Methylation of DNA purines 4 hr after a single i.v. injection (0.017 mmol/kg) of [methyl-14C]MBN was highest in the esophagus (344 mumol 7-methylguanine per mol guanine), followed by liver, lung, and forestomach. Considerably less DNA methylation was produced by an equimolar dose of 4-MeMBN, with highest values in liver, followed by esophagus (22 mumol 7-methylguanine per mol guanine) and lung. However, s.c. injections of equitoxic doses of MBN (18 mg/kg) and 4-MeMBN (394 mg/kg) produced similar amounts of 7-methylguanine in esophageal nucleic acids. These data indicate that the lower toxicity and carcinogenicity of 4-MeMBN after systemic administration are due to the rapid formation (mainly in the liver) and excretion via the urine of its benzoic acid derivative. The strong carcinogenic effect of orally administered 4-MeMBN can be explained by direct uptake from the drinking water into the esophageal mucosa. Following a single i.v. injection (0.017 mmol/kg) of [methylene-14C]MBN and 4-Me[methylene-14C]MBN, no benzylated bases were detectable in rat tissues. This indicates that the bioactivation of these compounds is initiated predominantly by hydroxylation at the methylene bridge leading to a methylating rather than a benzylating intermediate as the ultimate carcinogen.

The metabolic activation of chrysene by hamster embryo cells.

The major deoxyribonucleoside--hydrocarbon adducts present in hydrolysates of DNA isolated from hamster embryo cells treated with chrysene were examined by chromatography on Sephadex LH20 and by h.p.l.c. on Zorbax ODS. The results show that both major adducts have chromatographic properties identical to those of adducts formed when r-1,t-2-dihydroxy-t-3,4-oxy-1,2,3,4-tetrahydrochrysene reacts with DNA and provide evidence that metabolic activation of chrysene occurs via the formation of this 'bay-region' diol-epoxide.

DNA methylation by N-nitrosomethylbenzylamine in target and non-target tissues of NMRI mice.

Adult female NMRI mice received a single i.p injection of N-nitroso(methyl-14C)methylbenzylamine (2.5 mg/kg body weight). Multiple weekly applications of such a dose, by this route, have previously been shown to induce lung adenomas and forestomach carcinomas in all experimental animals. After a survival time of 6 h, DNA was isolated from various tissues and analysed for methylated purines by separation of the acid hydrolysate on Sephasorb columns. Highest concentrations of 7-methylguanine and 06-methylguanine were present in hepatic DNA, followed by lung and forestomach. DNA methylation in the oesophagus was only 21% less than in forestomach. Since both tissues develop a high tumour incidence after oral administration of N-nitrosomethylbenzylamine (MBN), this observation suggests that despite their anatomical similarities the level of DNA modification required for malignant transformation differs considerably in these tissue. In the remaining organs, DNA alkylation was either considerably less (colon, glandular stomach, kidney) or not detectable (small intestine, spleen). These date indicate that following i.p. injection in mice, MBN is preferentially metabolised in a non-target organ (liver). Among the various other tissues investigated, highest levels of initial DNA methylation were present in forestomach and lung, i.e., the principal target organs of MBN for this route of application.

Preferential methylation of target organ DNA by the oesophageal carcinogen N-nitrosomethylbenzylamine.

Male Wistar rats received a single i.v. injection of the oesophageal carcinogen N-nitroso[methyl-14C]-methylbenzylnitrosamine (2.5 mg/kg body weight). Rapid distribution of the carcinogen occurred, with highest initial concentrations in liver and kidney. Within 10 min after the injection, 14C-labelled metabolites accounted for 50% of the total radioactivity present in the oesophagus, for approximately 25% in liver and forestomach, and for less than 20% in all other organs investigated. Decay of the carcinogen in rat serum followed first-order kinetics with a half-life of 35 min. Of the total radioactivity administered, 49% was exhaled as 14CO2 within 10 h and an additional 5-10% was excreted via urine and faeces. Four hours after a single i.v. injection of N-nitroso-[methyl-14C]benzylnitrosamine methylation of purine bases in DNA was most extensive in the oesophagus, followed by liver, lung and forestomach DNA. In the remaining tissues, DNA methylation was either considerably less (kidney, glandular stomach, spleen) or not at all detectable (ileum, colon, brain). At this time the concentration of the promutagenic base O6-methylguanine in oesophageal DNA was six times higher than in lung and nine times higher than in hepatic DNA. These data suggest that in the rat the selective induction of oesophageal tumours by N-nitrosomethylbenzylamine and related asymmetrical nitrosamines is mediated by a preferential bioactivation of the carcinogen in the target organ.

Elimination and metabolism of dimethylnitrosamine (DMN) by Xenopus laevis and other amphibians.

The elimination of (14C)-DMN after i.p. injection into Xenopus was measured, as was the metabolism in vitro of (14C)-DMN by liver from Xenopus and 9 other amphibian species. In view of its rapid elimination from the body and low rate of metabolism by Xenopus liver in vitro, DMN is unlikely to be toxic or carcinogenic in Xenopus.

The effects of alpha and beta adrenergic agents on spleen cell antigen binding in four amphibian species.

Adults of two urodele amphibian species (Triturus cristatus carnifex and Cynops hongkongensis) and two anuran species (Rana temporaria and Xenopus laevis laevis) were immunized with a 25% suspension of sheep or horse erythrocytes. After eight or 14 days, splenic lymphocytes were removed, and their specific red cell-binding capacities tested by immunocytoadherence. Antigen-binding cells were classified as high-dose nonsecretory (S-) or secretory (S+), according to whether they bound a single layer or several layers or erythrocytes. The stimulation of both alpha and beta adrenoreceptors reduced the numbers of S+ rosettes formed by Triturus and Cynops lymphocytes, whereas a beta agonist increased and an alpha agonist decreased S+ rosette formation by Rana and Xenopus splenic lymphocytes. These effects were blocked by alpha and beta adrenoreceptor antagonists. Low-dose immunization of Xenopus with a 0.0025% suspension of sheep erythrocytes gave a minimal number of S+ rosettes two and eight days after immunization, and beta adrenoreceptor stimulation had no effect on antigen binding. These results are discussed in terms of the distribution of alpha and beta adrenoreceptors in amphibians and possible relationships between S+ and high-dose S- antigen-binding cells, and support the view that functional lymphocyte heterogeneity exists in these lower vertebrates.

Improved continuous wave frequency modulated sonars with aural displays.

This paper discusses methods for reducing the effects of the reset hiatus and wavelength related variations in received signal strength on the aural displays produced by simple continuous wave frequency modulated sonars. Two techniques that have been developed for reducing the effects of signal phase and amplitude discontinuities are described. As a practical example of the improved performance afforded by one of these techniques, a novel short range sonar for examining cardiovascular structures is discussed in detail.