Ulf Rannug

The mutagenic effect of 1,2-dichloroethane on Salmonella typhimurium I. Activation through conjugation with glutathion in vitro

One of the main components in the waste products from vinyl chloride industries (EDC-tar), is ethylene dichloride (1,2-dichloroethane). This compound has been tested for mutagenicity on Salmonella typhimurium TA 1535. It is concluded that 1,2-dichloroethane gives a weak direct mutagenic effect, which is enhanced by addition of the postmitochondrial liver fraction (S-9). This activation is NADPH-independent and non microsomal. It is caused by a factor in the soluble fraction (115 000 g supernatant). This activation was further enhanced by the addition of glutathione but not by the addition of L-cysteine, N-acetyl-L-cysteine or 2-mercaptoethanol. No activation was observed when glutathione was added in the presence of a totally denaturated S-9 fraction or in the absence of this fraction. Activation of 1,2-dichloroethane was also found in the presence of glutathione and glutathione S-transferase A and C but not with glutathione S-tranferase B. A synthetic conjugate S-(2-chloroethyl)-L-cysteine gave a strong direct mutagenic effect at concentrations where no effects were seen with 1,2-dichloroethane. It is thus concluded that 1,2-dichloroethane is activated by conjugation to glutathione. Another main component in EDC-tar, 1,1,2-trichloroethane, was not mutagenic under any of our experimental conditions. For comparison 1,2-dibromoethane was also tested and gave a stronger direct mutagenic effect than 1,2-dichloroethane. Like the latter 1,2-dibromoethane was also activated by a NADPH-independent process.

The suggested physiologic aryl hydrocarbon receptor activator and cytochrome P4501 substrate 6-formylindolo[3,2-b]carbazole is present in humans

Dioxins and other polycyclic aromatic compounds formed during the combustion of waste and fossil fuels represent a risk to human health, as well as to the well being of our environment. Compounds of this nature exert carcinogenic and endocrine-disrupting effects in experimental animals by binding to the orphan aryl hydrocarbon receptor (AhR). Understanding the mechanism of action of these pollutants, as well as the physiological role(s) of the AhR, requires identification of the endogenous ligand(s) of this receptor. We reported earlier that activation of AhR by ultraviolet radiation is mediated by the chromophoric amino acid tryptophan (Trp), and we suggested that a new class of compounds derived from Trp, in particular 6-formylindolo[3,2-b]carbazole (FICZ), acts as natural high affinity ligands for this receptor. Here we describe seven new FICZ-derived indolo[3,2-b]carbazole-6-carboxylic acid metabolites and two sulfoconjugates, and we demonstrate the following. (i) FICZ is formed efficiently by photolysis of Trp upon exposure to visible light. (ii) FICZ is an exceptionally good substrate for cytochromes P450 (CYP) 1A1, 1A2, and 1B1, and its hydroxylated metabolites are remarkably good substrates for the sulfotransferases (SULTs) 1A1, 1A2, 1B1, and 1E1. Finally, (iii) sulfoconjugates of phenolic metabolites of FICZ are present in human urine. Our findings indicate that formylindolo[3,2-b]carbazols are the most potent naturally occurring activators of the AhR signaling pathway and may be the key substrates of the CYP1 and SULT1 families of enzymes. These conclusions contradict the widespread view that xenobiotic compounds are the major AhR ligands and CYP1 substrates.

The mutagenicity of chloroethylene oxide, chloroacetaldehyde, 2-chloroethanol and chloroacetic acid, conceivable metabolites of vinyl chloride

Previous investigations have shown that the carcinogen vinyl chloride causes base-pair substitution in the bacterium Salmonella typhimurium. The ability of four conceivable metabolites-chloroethylene oxide, chloroacetaldehyde, 2-chloroethanol and chloroacetic acid-to cause base-pair substitution directly in Salmonella typhimurium TA1535 has been compared. The main comparison was performed at initial concentrations from 0.1 to 1.5 mM. In this region, however, a mutagenic effect was observed only with chloroethylene oxide and chloroacetaldehyde, the former being approximately 20 times more effective than the aldehyde when compared on a molar basis.2-Chloroethanol and chloroacetic acid were studied also at higher concentration (1 mM-1 M), and a weak mutagenic response was found with 1 M 2-chloroethanol solution. With chloroacetic acid no enhancement of the mutation frequency could be detected. Chloroethylene oxide was found to be approximately 450 times more effective as a mutagen than chloroacetaldehyde when the comparison is based on exposure doses, defined as the time-dependent concentrations of the compounds in the treatment solutions, integrated between the times of onset and termination of treatment. Similarly, chloroethylene oxide was 10,000-15,000 times more effective as a mutagen than ethylene oxide, used as a positive control.

Rapid and transient induction of CYP1A1 gene expression in human cells by the tryptophan photoproduct 6-formylindolo[3,2-b]carbazole.

Studies to assess the induction of CYP1A1 gene expression by tryptophan derived oxidation products which are suggested as endogenous ligands for the Ah receptor are described. For the two high affinity Ah receptor ligands produced from tryptophan, the chemical structure was recently identified as 6-formylindolo[3,2-b]carbazole (FICZ) and 6,12-diformylindolo[3,2-b]carbazole (dFICZ), respectively. Therefore these two compounds show a close similarity to the indolecarbinol-derived condensation product indolo[3,2-b]carbazole (ICZ). Incubation of cells from a human keratinocyte (HaCaT) cell line together with ICZ, FICZ, dFICZ and some structurally related indole compounds was performed. The compound with the highest affinity to the Ah receptor, FICZ, was found to be the most efficient inducer of CYP1A1 gene expression in short time incubation (0.5 h) experiments. With longer incubation times (24 h) ICZ was the most efficient inducer. The two most active compounds, FICZ and ICZ, caused increased mRNA levels already at a concentration of 100 pM. FICZ was also shown to increase CYP1A1 mRNA levels in fresh human peripheral blood cells at the same low concentration. FICZ and ICZ were furthermore compared with regard to their capacity to inhibit cDNA-expressed human CYP1A1 enzyme and FICZ was found to be the most potent inhibitor. The inhibition was, however, transient in character indicating that FICZ is also an exceptionally good substrate for the CYP1A1 enzyme. The results showing the potent and transient effect of these formylindolocarbazoles, thus emphasize their important properties as signal substances in the Ah receptor pathway. This makes the most potent compound, FICZ, a good candidate for the endogenous ligand of the Ah receptor necessary for normal development and for the basal expression of Ah receptor-dependent genes.

Effect of fuel polycyclic aromatic hydrocarbon content on the emissions of polycyclic aromatic hydrocarbons and other mutagenic substances from a gasoline-fueled automobile

Exhaust emissions from a fuel-injected, Otto engine equipped car driven according to the US Federal Test Procedure (FTP-73) were characterized chemically and by mutagenicity tests. Four fuel qualities of different polycyclic aromatic hydrocarbon (PAH) content were used. Emission variables investigated were regulated pollutants (CO, unburned hydrocarbons, NO/sub x/), particle emissions, emission of particle and gas phase associated polycyclic aromatic hydrocarbons, aldehyde emissions, emissions of benzene, toluene, ethene, and propene, and mutagenicity in particle and gas phases. The amount of formed PAH emitted in the exhaust is approximately constant. In fuels with low PAH contents a large proportion of emitted PAH was formed. Most of the cyclopenta(cd)pyrene and benzo(b and k) fluoranthene, greater than or equal to 80% and greater than or equal to 70%, respectively, is formed in the combustion process. All the exhaust samples, except one, in both the particulate phase and the gas phase, gave significant mutagenic effects on Salmonella typhimurium tester strains TA98 and TA100 with or without a metabolizing system (S9). Generally, the gas phase gave a higher effect compared to the particulate phase. The best correlation between emitted PAH and mutagenicity was found for the particulate phase and TA100+S9.

Inhibition of cytochrome P4501-dependent clearance of the endogenous agonist FICZ as a mechanism for activation of the aryl hydrocarbon receptor

Altered systemic levels of 6-formylindolo[3,2-b]carbazole (FICZ), an enigmatic endogenous ligand for the aryl hydrocarbon receptor (AHR), may explain adverse physiological responses evoked by small natural and anthropogenic molecules as well as by oxidative stress and light. We demonstrate here that several different chemical compounds can inhibit the metabolism of FICZ, thereby disrupting the autoregulatory feedback control of cytochrome P4501 systems and other proteins whose expression is regulated by AHR. FICZ is both the most tightly bound endogenous agonist for the AHR and an ideal substrate for cytochrome CYP1A1/1A2 and 1B1, thereby also participating in an autoregulatory loop that keeps its own steady-state concentration low. At very low concentrations FICZ influences circadian rhythms, responses to UV light, homeostasis associated with pro- and anti-inflammatory processes, and genomic stability. Here, we demonstrate that, if its metabolic clearance is compromised, femtomolar background levels of this compound in cell-culture medium are sufficient to up-regulate CYP1A1 mRNA and enzyme activity. The oxidants UVB irradiation and hydrogen peroxide and the model AHR antagonist 3′-methoxy-4′-nitroflavone all inhibited induction of CYP1A1 enzyme activity by FICZ or 2,3,7,8-tetrachlorodibenzo-p-dioxin, thereby subsequently elevating intracellular levels of FICZ and activating AHR. Taken together, these findings support an indirect mechanism of AHR activation, indicating that AHR activation by molecules with low affinity actually may reflect inhibition of FICZ metabolism and raising questions about the reported promiscuity of the AHR. Accordingly, we propose that prolonged induction of AHR activity through inhibition of CYP1 disturbs feedback regulation of FICZ levels, with potential detrimental consequences.

UV-induced CYP1A1 gene expression in human cells is mediated by tryptophan.

Induction of cytochrome P-4501A1 (CYP1A1) activity by UV has been observed earlier in animal studies via a mechanism that has not yet been resolved. Our previous data have indicated that formylated indolocarbazoles which are formed by UV irradiation of tryptophan solutions are very potent Ah-receptor agonists. To evaluate the effect of UV light on cytochrome P4501A1 gene expression, we studied the induction of CYP1A1 mRNA by UV irradiation of cultured human keratinocytes (HaCaT cell line), primary human blood lymphocytes and mouse Hepa-1 cells. The cells were exposed to UV light delivered by a bank of 6 Philips TL20/12RS sun lamps emitting primarily in the UVB range in the absence and presence of tryptophan. A semiquantitative reverse transcriptase-linked polymerase chain reaction (RT-PCR) was used for analysis of gene expression in the treated cells. The results show that the CYP1A1 mRNA level induced by UV in the presence of tryptophan was higher than that induced by UV alone in both HaCaT cells and lymphocytes after 3 h of incubation post-UV irradiation. To find out if the induction by UV light is caused by the formation of an Ah receptor ligand, Hepa-1 wild-type and Ah receptor deficient c12 cell lines were applied. Wild-type (wt) cells were inducible either by the tryptophan photoproduct 6-formylindolo[3,2-b]carbazole (FICZ) or by UV-irradiation but very low or undetectable levels were observed in the c12 cells. This shows that the induction of gene expression by FICZ and UV is Ah receptor dependent. Together, these results indicate that UV-induced CYP1A1 gene expression in mammalian cells is mediated by an Ah receptor ligand formed from tryptophan. Thus, the photoproducts of tryptophan are suggested to be mediators of light via binding to the Ah receptor and as such also could have a role in light-regulated biological rhythms.

The mutagenic effect of 1,2-dichloroethane on Salmonella typhimurium. II. activation by the isolated perfused rat liver

In this investigation Salmonella typhimurium strain TA 1530 and TA 1535 were combined with isolated perfused rat liver. Samples of perfusate and bile produced were tested for mutagenicity after treatment with 1,2-dichloroethane (DCE), 1,2-dibromoethane (DBE) or 2-chloroethanol. The results are in good agreement with our previous experiments which indicate that both DEC and DBE are activated through conjugation with glutathione (GSH). Most GSH conjugates are normally excreted in bile. Following liver perfusion the bile was highly mutagenic after DCE and DBE treatments, while 2-chloroethanol did not have this effect. The highest mutagenic effect was seen 15--30 min after the addition of DCE or DBE. The production of mutagenic bile also occurred in mice treated in vivo with DCE. One possible metabolic endproduct of a GSH conjugate is the corresponding mercapturic acid. Thus synthetic N-acetyl-S-(2-chloroethyl)-L-cysteine was tested on TA 1535 and found to be as mutagenic as S-(2-chloroethyl)-L-cysteine in the concentration range 0.2--0.6 mumol/plate. Differences and similarities in the metabolism of DCE and vinyl chloride are discussed on the basis of these results.

Mutagenicity and metabolism studies on 12 thiuram and dithiocarbamate compounds used as accelerators in the Swedish rubber industry.

12 thiuram and dithiocarbamate compounds used in the rubber industry as accelerators, and to some extent as sources of sulfur, were tested, as well as carbon disulfide, a metabolite found in vivo after dithiocarbamate treatment, for mutagenicity in Salmonella typhimurium. A mutagenic effect on the base-substitution-sensitive strains TA1535 and TA100 was found for 7 compounds. The most potent directly acting mutagens were: tetramethylthiuram disulfide (TMTD), zinc dimethyldithiocarbamate (ziram), cadmium diethyldithiocarbamate and zinc diethyldithiocarbamate. Tetraethylthiuram disulfide (TETD), also known as Antabus, and carbon disulfide were non-mutagenic. The relatively low direct mutagenic effect of tetramethylthiuram monosulfide (TMTM) was enhanced in the presence of a metabolizing system (S9 mix). A hypothesis is given regarding the activation process of the monosulfide TMTM.

Quercetin, Resveratrol, and Curcumin Are Indirect Activators of the Aryl Hydrocarbon Receptor (AHR)

Several polyphenols have been shown to activate the aryl hydrocarbon receptor (AHR) in spite of the fact that they bind to the receptor with low affinity. The aim of this study was to investigate whether quercetin (QUE), resveratrol (RES), and curcumin (CUR) interfere with the metabolic degradation of the suggested endogenous AHR ligand 6-formylindolo[3,2-b]carbazole (FICZ) and thereby indirectly activate the AHR. Using recombinant human enzyme, we confirmed earlier reported inhibitory effects of the polyphenols on cytochrome P4501A1 (CYP1A1) activity, and inhibition of metabolic clearance of FICZ was documented in FICZ-treated immortalized human keratinocytes (HaCaT). CYP1A1 activity was induced in HaCaT cells by all three compounds, and when they were added together with FICZ, a prolonged activation was observed after a dose-dependent inhibition period. The same pattern of responses was seen at the transcriptional level as determined with a CYP1A1 reporter assay in human liver hepatoma (HepG2) cells. To test the ability of the polyphenols to activate the AHR in the absence of FICZ, the cells were treated in medium, which in contrast to commercial batches of medium did not contain background levels of FICZ. Importantly, AHR activation was only observed in the commercial medium. Taken together, these findings suggest that QUE, RES, and CUR induce CYP1A1 in an indirect manner by inhibiting the metabolic turnover of FICZ. Humans are exposed to these compounds through the diet and nutritional supplements, and we propose that altered systemic levels of FICZ caused by such compounds may have physiological consequences.