Comparison of rat and guinea pig as sources of the S9 fraction in the salmonella/mammalian microsome mutagenicity test

A highly significant enhancement of mutagenicity occurs with 11 polycyclic aromatic hydrocarbons when 3-methylcholanthrene-induced guinea pig liver S9 is substituted for Aroclor-induced rat liver S9 in the Ames test. The use of MC-induced guinea pig liver S9 is particularly valuable for detecting the weak mutagenicity of benz[c]acridine, which is barely positive in a standard Ames assay. However, anthracene and phenanthrene, which are generally considered not to be carcinogens, remain non-mutagenic for strain TA100. This enhancement of mutagenicity does not correlate with arylhydrocarbon hydroxylase activities of the various liver preparations and does not apply to certain other non-PAH mutagens, including β-naphthylamine, aflatoxin B₁ and 4-dimethylaminoazobenzene.     

Activation of aromatic amines to mutagens by various animal species including man

2-Acetylaminofluorene, 2-aminofluorene, 4-aminobiphenyl, 2-naphthylamine, 2-aminoanthracene and benzidine were assayed for mutagenicity in the Ames test in the presence of hepatic microsomal preparations derived from mouse, hamster, rat, pig and man. Prior to each mutagenicity assay all activation systems were fully characterized with respect to mono-oxygenase and mixed-function amine oxidase activities. All compounds were metabolically activated to mutagens by all activation systems, but with markedly different efficiencies, hamster being the only species which readily activated all amines. The hamster also exhibited the highest ethoxyresorufin O-deethylase and dimethylaniline N-oxidase activities.     

Oxidation of Anthracene and Benzo[a]pyrene by Laccases from Trametes versicolor

The in vitro oxidation of the two polycyclic aromatic hydrocarbons anthracene and benzo[a]pyrene, which have ionization potentials of <=7.45 eV, is catalyzed by laccases from Trametes versicolor. Crude laccase preparations were able to oxidize both anthracene and the potent carcinogen benzo[a]pyrene. Oxidation of benzo[a]pyrene was enhanced by the addition of the cooxidant 2,2(prm1)-azinobis(3-ethylbenzthiazoline-6-sulfonate) (ABTS), while an increased anthracene oxidizing ability was observed in the presence of the low-molecular-weight culture fluid ultrafiltrate. Two purified laccase isozymes from T. versicolor were found to have similar oxidative activities towards anthracene and benzo[a]pyrene. Oxidation of anthracene by the purified isozymes was enhanced in the presence of ABTS, while ABTS was essential for the oxidation of benzo[a]pyrene. In all cases anthraquinone was identified as the major end product of anthracene oxidation. These findings indicate that laccases may have a role in the oxidation of polycyclic aromatic hydrocarbons by white rot fungi.     

Mutagenicity of benzo[a]pyrene 7,8-dihydrodiol and 7,12-dimethylbenz[a]anthracene 3,4-dihydrodiol in S. typhimurium mediated by microsomes from rat liver and mouse skin

The mutagenic activities of trans-7,8-dihydro-7,8-dihydroxybenzo[a]-pyrene (BP 7,8-diol) and of trans-3,4-dihydroxy-7,12-dimethylbenz[a]-anthracene (DMBA 3,4-diol) towards S. typhimurium TA100 were measured in assays that were carried out on a micro-scale in liquid medium in the presence of microsomal fractions prepared from mouse skin or rat liver. In the presence of an NADPH-generating system, microsomal enzymes converted both diols into mutagens that were probably the respective 'bay-region' diol-epoxides. The rate of the enzyme-catalysed conversion of the BP 7,8-diol into mutagens by microsomal preparations from mouse epidermis was similar to that occurring with microsomes from rat liver. Pretreatment of mice by the topical application of benz[a]anthracene (BA) or 7,12-dimethylbenz[a]-anthracene (DMBA) increased the mutagenic activity of BP 7,8-diol mediated by mouse skin microsomal preparations by 2-fold and this was paralleled by a 4-fold increase in epidermal aryl hydrocarbon (benzo[a]pyrene) hydroxylase (AHH) activity. The results are discussed in relation to the high susceptibility of mouse skin to polycyclic aromatic hydrocarbon (PAH) carcinogenesis.     

Induction of sister-chromatid exchanges in Chinese hamster ovary cells treated in vitro with non-k-region dihydrodiols of 7-methylbenz[a]anthracene and benzo[a]pyrene

Studies were carried out on the incidence of sister-chromatid exchanges induced in Chinese hamster ovary cells by in vitro treatment with the polycyclic aromatic hydrocarbons 7-methylbenz[a]anthracene and benzo[a]pyrene and with related K-region and non-K-region dihydrodiols. Appreciable increases in the incidence of sister-chromatid exchanges were apparent in cells treated with non-K-region dihydrodiols: the most active compounds were 3,4-dihydro-3,4-dihydroxy-7-methylbenz[a]anthracene and 7,8-dihydro-7,8-dihydroxybenzo[a]pyrene and the effects were dose-dependent. The parent hydrocarbons and the related K-region dihydrodiols induced some sister-chromatid exchanges but they were considerably less active than these two non-K-region diols. The results suggest that this system may usefully be applied to studies aimed at determining which dihydrodiols are important in the metabolic activation of the carcinogenic polycyclic hydrocarbons. These and other results also infer that Chinese hamster ovary cells possess some intrinsic ability to metabolize such compounds in the absence of exogenous activation systems.     

Promutagen activation by rodent-liver postmitochondrial fractions in the L5178Y/TK cell mutation assay

Individual S9 microsomal fractions prepared from normal livers of 8 rodent species or strains and from 1 rat strain pretreated with Aroclor 1254, were used to metabolize the promutagens N-acetyl-2-aminofluorene, 1,2--benzanthracene, to metabolize the promutagens N-acetyl-2-aminofluorene, 1,2-benzanthracene, benzo[a]pyrene, and 3-methylcholanthrene to active forms during 3-h co-incubation in the presence of L5178Y/TK^+/− cells. The 8 compatible S9 preparations all converted each of the 4 chemical carcinogens into active mutagens with varied efficiencies except for the Aroclor-induced rat S9/benzanthracene combination which produced only weak activity. Aroclor induction did not notably enhance the mutagenicity of benzo[a]pyrene or 3-methylcholanthrene beyond that activity mediated by the other non-induced preparations. Syrian hamster S9 and, to a lesser degree, C57BL/6J mouse S9 were exceptionally active in converting N-acetyl-2-aminofluorene to toxic and mutagenic metabolites. One source of Swiss mouse liver (Blu : Ha ICR) provided the most active S9 when tested with the 3 polycyclic aromatic hydrocarbons.In general, mutagenicity and cytotoxicity were roughly correlated within S9 + promutagen combinations. Almost all of the methylcholanthrene metabolizing activity was lost by the 12th week when Aroclor-induced rat S9 was held at −20°C, yet this activity remained constant when similar S9 was stored at −80°C for 14 weeks. Surprisingly, some S9 sources including the induced rat-liver preparation converted anthracene to a weak or border-line mutagen. The activation of both 1,2-benzanthracene and anthracene may be linked within each species or strain although Aroclor induction enhanced anthracene mutagenicity yet attenuated the mutagenicity of 1,2-benzanthracene. Collectively, these data underscore the current inchoate state of development for S9 coupled somatic cell mutation assays.     

Enzymatic characterization of the polynuclear aromatic hydrocarbons activating rat-liver preparations used in the mutagenicity test of Ames

Stability studies were performed on the mono-oxygenase system involved, in particular, in the activation of polynuclear aromatic hydrocarbons (PAHs) present in rat-liver preparations used in the Ames mutagenicity test. The results indicated a good stability of the spectral response of the cytochrome-P-450 system, but a much lower stability of its enzymatic activities measured with various substrates, thus showing the inadequacy of the spectral response to characterize the PAH mono-oxygenase activity of the preparations. Epoxide hydrolase activity was found to be stable. Various mono-oxygenase activities were measured in preparations induced with phenobarbital, 3-methylcholanthrene or Aroclor 1254. The activities of two enzymes, benzo[a]pyrene hydroxylase and ethoxyresorufin-O-dealkylase, were found suitable to characterize the capacity of the preparations to metabolize PAH to mutagens. The efficiency of the same preparations to promote the mutagenicity of benzo[a]pyrene and aflatoxin B1 in the Ames test was determined. There was an excellent general correlation between the efficiencies for mutagenic activation of the preparations and the two enzymatic activities mentioned above. Determination of ethoxyresorufin-O-dealkylase (or benzo[a]pyrene hydroxylase) and benzo[a]pyrene 4,5-oxide hydrolase activities is proposed for characterizing the rat-liver preparations used in the Ames test.     

Profiles of polycyclic aromatic hydrocarbon metabolites after treatment with various inducers of microsomal rat liver monoxygenases (author's transl)

The microsomal oxidation of 12 frequently occurring environmental polycyclic aromatic hydrocarbons after incubation with rat-liver microsomes has been studied and their metabolites characterized by means of gas-liquid chromatography/mass spectrometry. The method enables the detection and characterisation of phenols, diols, triols, and tetrols as trimethylsilyl ethers beside the original hydrocarbons. Moreover, the induction properties of some carcinogenic and non-carcinogenic hydrocarbons (benz[a]anthracene, pyrene, chrysene, benzo[a]-pyrene, benzo[e]pyrene, benzo[b]fluoranthene, benzo[j]fluoranthene, benzo[k]fluoranthene) have been studied. Except pyrene and benzo[e]pyrene, all compounds investigated significant but different induction factors. The relevance of the induction for an estimation of the biological effect of environmental polycyclic aromatic hydrocarbons is discussed.     

Fungal metabolism and detoxification of polycyclic aromatic hydrocarbons

The mutagenic activity of ethyl acetate extracts of culture medium from Cunninghamella elegans incubated 72 h with various polycyclic aromatic hydrocarbons (PAHs) was evaluated in the Salmonella typhimurium reversion assay. All of the PAH extracts were assayed in tester strains TA98 and TA100 both with and without metabolic activation using a liver fraction from Aroclor 1254-treated rats. None of the extracts from fungal incubations with the mutagenic PAHs, benzo[a]pyrene, 7,12-dimethylbenz[a]anthracene, 3-methylcholanthrene and benz[a]anthracene, as well as the non-mutagenic PAHs, naphthalene, phenanthrene and anthracene, displayed any appreciable mutagenic activity. In addition, time course experiments indicated that the rate of decrease in mutagenic activity in the extracts from cultures incubated with benzo[a]pyrene or 7,12-dimethylbenz[a]anthracene was coincident with the rate of increase in total metabolism. The results demonstrated the ability of the fungus C. elegans to detoxify known carcinogens and mutagens and suggests that this organism may play an important role in the metabolism and inactivation of PAHs in the environment.Abbreviations hplc high performance liquid chromatography - tlc thin layer chromatography - PAH polycyclic aromatic hydrocarbon     

Hamster hepatocyte-mediated activation of procarcinogens to mutagens in the L5178Y/TK mutation assay

Eight procarcinogens including three nitrosamines, three polycyclic hydrocarbons, and two aromatic amines were tested for mutagenic potential at the thymidine kinase (TK) locus in L5178Y mouse lymphoma cells co-cultivated with viable hamster hepatocytes. All eight chemicals produced substantial mutagenic activity as indicated by increased trifluorothymidine resistance in L5178Y cells treated in the presence of hepatocytes. Mutagenic responses to benzo[a]pyrene, 3-methyl-cholanthrene, N-nitrosodiethylamine, and N-nitrosodipropylamine first increased, then plateaued within the range of mutagen concentrations tested, while consistent dose-dependent increases in mutant frequencies were observed following 2-aminoanthracene, 2-aminofluorene, or N-nitrosodimethylamine treatments. The relatively flat portions of the mutant frequency curves for benzo[a]pyrene and 3-methylcholanthrene coincided with maximum chemical solubility as obvious from visible or microscopically detectable precipitate. These hamster cells readily facilitated the metabolism of 1,2-benzanthracene to a detectable mutagen and were especially competent in the activation of the two aromatic amines. Thus, cultured hamster hepatocytes can activate a variety of chemical carcinogens including polycyclic hydrocarbons to mutagens in a whole cell-mediated in vitro assay using L5178Y/TK^+/? cells as the target organism.     

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.     

Sister-chromatid exchange induction by polycyclic aromatic hydrocarbons in an intact cell system of adult rat-liver epithelial cells

Adult rat-liver epithelial cell lines possess intrinsic metabolic capability for the biotransformation of xenobiotics and thus, are sensitive to a broad spectrum of mutagens/carcinogens in a mutagenesis assay at the hypoxanthine-guanine phosphoribosyl transferase locus. To provide another end-point of biological significance in these lines, we have investigated the application of adult rat-liver epithelial cell line 18 in a sister-chromatid exchange assay. Significant dose-dependent increases in the sister-chromatid exchange frequency occurred when liver cells were exposed to benzo[a]pyrene and 7,12-dimethylbenz[a]anthracene. A weak but positive response was elicited by benz[a]anthracene. The present observations thus confirm the capacity of these cells to generate genotoxic metabolites from activation-dependent mutagens/carcinogens and indicate a relationship between the production of mutations and sister-chromatid exchanges by polycyclic aromatic hydrocarbons.     

Microbial degradation and detoxification of high molecular weight polycyclic aromatic hydrocarbons by Stenotrophomonas maltophilia strain VUN 10,003

The ability of Stenotrophomonas maltophilia strain VUN 10,003 to degrade and detoxify high molecular weight polycyclic aromatic hydrocarbons (PAHs) was evaluated in a basal liquid medium. Using high cell density inocula of strain VUN 10,003, the concentration of pyrene, fluoranthene, benz[a]anthracene, benzo[a]pyrene, dibenz[a, h]anthracene and coronene decreased by 98, 45, 26, 22, 22 and 55% over periods ranging from 5 to 42 d. When a PAH mixture containing three- to seven-ring compounds was used, degradation of both low and high molecular weight compounds occurred concurrently. Mutagenicity assays (Ames Test) demonstrated a decrease in the mutagenic potential of dichloromethane culture extracts from all cultures containing single PAH over the incubation period, corresponding to the decrease in the concentration of the PAH. These observations indicate that strain VUN 10,003 could be used for the detoxification of PAH-contaminated wastes.     

The homogeneity of rat liver microsomal cytochrome P-448 activity and its role in the activation of benzo[a]pyrene to mutagens

The O-deethylation of ethoxyresorufin and the metabolic activation of benzo[a]pyrene to mutagens were determined in hepatic microsomal preparations from control and induced animals. An excellent direct correlation (r = 0.95) has been observed between ethoxyresorufin O-deethylase and the metabolic activation of benzo[a]pyrene to mutagens when the fraction of cytochromes P-450 present as cytochrome P-448 was altered by the administration of phenobarbitone and 3-methylcholanthrene alone or in combination with 9-hydroxyellipticine. The correlation between these activities was maintained following treatment of animals with Arochlor 1254, benzo[a]pyrene, benzo[e]pyrene, 7,12-dimethylbenzo[a]anthracene,2-anthramine and 2-naphthylamine.     

The presence of the mutagenic polycyclic aromatic hydrocarbons benzo[a]pyrene and benz[a]anthracene in creosote P1

Several fractions of creosote P1 separated by TLC showed mutagenicity towards Salmonella typhimurium TA98. Thus mutagenicity is probably caused by the presence of mutagenic aromatic hydrocarbons. The mutagenic polycyclic aromatic hydrocarbons, benzo[a]pyrene and benz[a]anthracene, were detected in concentrations of 0.18 and 1.1% respectively. Because these compounds are probably not essential for the wood-preserving properties of creosote , a more selective composition of the product should be considered.     

Selective oxidation of mitochondrial glutathione in cultured rat adrenal cells and its relation to polycyclic aromatic hydrocarbon-induced cytotoxicity

Primary cultures of rat adrenal cells, as well as rat adrenals in vivo, are sensitive to the potent carcinogen 7,12-dimethylbenz[a]anthracene and its liver metabolite 7-hydroxymethyl-12-methylbenz[a]anthracene, whereas unmethylated polycyclic aromatic hydrocarbons like benzo[a]pyrene or benzo[a]anthracene are ineffective. The adrenocorticolytic potencies of the hydrocarbons are affected by adrenocorticotrophic hormone and various steroids, cytochrome P450 inhibitors, and antioxidants. In the present investigation digitonin was used to fractionate cultured rat adrenal cells. It was found that the mitochondria and cytosol of the cells contained 3-5 nmol/10(6) cells (approximately 15%) and 20-30 nmol/10(6) cells (approximately 85%) of the total soluble cellular glutathione equivalents, respectively. After exposing the cells to 7-hydroxymethyl-12-methylbenz[a]anthracene in the culture medium, a time- and concentration-dependent selective oxidation of mitochondrial glutathione was observed, whereas the effect on the cytosolic glutathione was negligible. Under the same conditions, 7,12-dimethylbenz[a]anthracene and benzo[a]pyrene were unable to alter the redox levels of the subcellular pools of glutathione. Omission of adrenocorticotrophic hormone lowered the oxidation of mitochondrial glutathione induced by 7-hydroxymethyl-12-methylbenz[a]anthracene about twofold. The results suggest that rat adrenal cells contain two separate pools of glutathione, one cytosolic and one mitochondrial, of which the latter is selectively influenced by 7-hydroxymethyl-12-methylbenz[a]anthracene. Moreover, it is concluded that rat adrenal cells offer a unique model system for general studies of the effects of a selective oxidation of mitochondrial glutathione on various cell functions. These effects may constitute early changes in cytotoxicity, preceding, e.g., membrane damage and loss of cytosolic components.     

Conversion of polycyclic aromatic hydrocarbons by <Emphasis Type="Italic">Sphingomonas</Emphasis> sp. VKM B-2434

A versatile bacterial strain able to convert polycyclic aromatic hydrocarbons (PAHs) was isolated, and a conversion by the isolate of both individual substances and PAH mixtures was investigated. The strain belonged to the Sphingomonas genus as determined on the basis of 16S rRNA analysis and was designated as VKM B-2434. The strain used naphthalene, acenaphthene, phenanthrene, anthracene and fluoranthene as a sole source of carbon and energy, and cometabolically oxidized fluorene, pyrene, benz[a]anthracene, chrysene and benzo[a]pyrene. Acenaphthene and fluoranthene were degraded by the strain via naphthalene-1,8-dicarboxylic acid and 3-hydroxyphthalic acid. Conversion of most other PAHs was confined to the cleavage of only one aromatic ring. The major oxidation products of naphthalene, phenanthrene, anthracene, chrysene, and benzo[a]pyrene were identified as salicylic acid, 1-hydroxy-2-naphthoic acid, 3-hydroxy-2-naphthoic acid, o-hydroxyphenanthroic acid and o-hydroxypyrenoic acid, respectively. Fluorene and pyrene were oxidized mainly to hydroxyfluorenone and dihydroxydihydropyrene, respectively. Oxidation of phenanthrene and anthracene to the corresponding hydroxynaphthoic acids occurred quantitatively. The strain converted phenanthrene, anthracene, fluoranthene and carbazole of coal-tar-pitch extract.     

Ah receptor in primate liver: binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin and carcinogenic aromatic hydrocarbons

Ah receptor in hepatic cytosols from adult cynomolgus monkeys (Macaca fasicularis) was identified and quantitated by its binding of the highly toxic chemical 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and the carcinogens 3-methylcholanthrene, benzo[a]pyrene, and dibenz[a,h]anthracene. The concentration of Ah receptor in cynomolgus hepatic cytosols (approximately 10 fmol/mg cytosol protein) was about one-quarter of that typically detected in rodent hepatic cytosols. Receptor concentrations were equal in male and female cynomolgus. [3H]TCDD bound to cytosolic receptor with high affinity (Kd approximately 3 nM). In rodents, Ah receptor is known to play a central role in toxicity caused by halogenated aromatic compounds and in carcinogenesis caused by polycyclic aromatic hydrocarbons. Existence of Ah receptor in monkeys indicates that the receptor also may mediate such responses in primates.     

Removal and transformation of high molecular weight polycyclic aromatic hydrocarbons in water by live and dead microalgae

The removal and transformation of seven high molecular weight polycyclic aromatic hydrocarbons (PAHs), namely benz[a]anthracene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene, dibenzo[a,h]anthracene, indeno[1,2,3-c,d]pyrene and benzo[g,h,i]perylene, by a freshwater microalga Selenastrum capricornutum under gold and white light irradiation was studied. The two light sources did not result in significant differences in the biodegradation of the selected PAHs in live algal cells, but white light was more effective in promoting photodegradation than was gold light in dead cells. The removal efficiency of seven PAHs, as well as the difference between live and dead microalgal cells, was PAH compound-dependent. Benz[a]anthracene and benzo[a]pyrene were highly transformed in live and dead algal cells, and dead cells displayed greater transformation levels than live cells. Further investigation comparing the transformation of single PAH compound, benzo[a]pyrene, by S. capricornutum and another green microalgal species, Chlorella sp., demonstrated that the transformation in dead cells was similar, indicating the process was algal-species independent. Dead algal cells most likely acted as a photosensitizer and accelerated the photodegradation of PAHs.