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Toxaphene (polychlorinated camphenes) is an insecticidal mixture of >670 chemicals, which was widely used until the mid 1980s. Due to their lipophilic and volatile nature, these chemicals accumulate in animal and human tissues and continue to be a major contaminant in marine and freshwater biota. Cytotoxic and genotoxic effects in mammalian test systems suggest that toxaphene is a carcinogen and reports support the hypothesis that toxaphene could have tumor-promoting potential in human breast tissue. In order to examine the potential of toxaphene as an environmental endocrine disrupter, we investigated its effect on the estrogen receptor (ER) function in human breast cancer MCF-7 cells. Using transient gene expression experiments, we observed approximately 60% and 80% inhibition of the constitutive and 17beta-estradiol induced ER-dependent transactivation, respectively. The involvement of the ER in the ability of toxaphene to block the estrogen action was verified by cotransfection studies in ER-negative MDA-MB-231 cells. The interference of toxaphene with the ER mediated responses was supported by a significant suppression of endogenously expressed pS2 RNA and decreased levels of secreted pS2 protein. These reproducible results indicate that toxaphene can disturb hormonal signals mediated by the ER and suggest that these environmental chemicals have potential endocrine disrupting activities which may affect the reproductive health and increase the risk of carcinogenesis. [ABSTRACT FROM PUBLISHER]

Bulky DNA adducts are biomarkers of exposure to aromatic compounds and of the ability of the individual to metabolically activate carcinogens and to repair DNA damage. Their ability to predict cancer onset is uncertain. We have performed a pooled analysis of three prospective studies on cancer risk in which bulky DNA adducts have been measured in blood samples collected from healthy subjects (N = 1947; average follow-up 51-137 months). In addition, we have performed a meta-analysis by identifying all articles on the same subject published up to the end of 2006, including case-control studies. In the pooled analysis, a weakly statistically significant increase in the risk of lung cancer was apparent (14% per unit standard deviation change in adduct levels, 95% confidence interval 1-28%; using the weighted mean difference method, 0.15 SD, units higher adducts in cases than in controls). The association was evident only in current smokers and was absent in former smokers. Also the meta-analysis, which included both lung and bladder cancers, showed a statistically significant association in current smokers, whereas the results in never smokers were equivocal; in former smokers, no association was detected. The results of our pooled and meta-analyses suggest that bulky DNA adducts are associated with lung cancer arising in current smokers after a follow-up of several years.

The aim of this study is to assess potential health risk of exposure to particle-associated polycyclic aromatic hydrocarbons (PAHs) in children living in a megacity with traffic congestion such as Bangkok. The study population comprised 184 Thai schoolboys (aged 8-13 years) attending schools adjacent to high-density traffic areas in Bangkok and schools located in the provincial area of Chonburi. The ambient concentration of total PAHs at roadsides in proximity to the Bangkok schools was 30-fold greater than at roadsides in proximity to the provincial schools (30.39 +/- 5.80 versus 1.50 +/- 0.28 ng/m(3); P < 0.001). Benzo(g,h,i)perylene (BghiP), an indicator of automobile exhaust emission, was the predominant PAH. Personal exposure to total PAHs and the corresponding benzo(a)pyrene (BaP) equivalent concentrations in Bangkok schoolchildren were 3.5-fold higher than in provincial schoolchildren (4.13 +/- 0.21 versus 1.18 +/- 0.09 ng/m(3); P < 0.001 and 1.50 +/- 0.12 versus 0.43 +/- 0.05 ng/m(3); P < 0.001, respectively). The concentration of urinary 1-hydroxypyrene (1-HOP) was significantly higher in Bangkok schoolchildren. Bulky carcinogen-DNA adduct levels in peripheral lymphocytes were also significantly higher (0.45 +/- 0.03 versus 0.09 +/- 0.00 adducts/10(8) nt; P < 0.001). Finally, a significantly higher level of DNA strand breaks and a significantly lower level of DNA repair capacity were observed in Bangkok schoolchildren (P < 0.001). This study indicates that Bangkok schoolchildren exposed to a high level of genotoxic PAHs in ambient air may be more vulnerable to the health impacts associated with the exposure to genotoxic pollutants than children in provincial areas and may have increased health risks for the development of certain diseases such as cancer.

Air pollution, containing high-level of ultrafine particles (UFP) and benzene, is a prominent environmental health problem in many cities of the World. We investigated the level of oxidative DNA damage in mononuclear blood cells (MNBC) by the comet assay as DNA strand breaks (SB) and formamidopyrimidine DNA glycosylase (FPG) sensitive sites in residents from three urban locations in Cotonou, Benin (taxi-moto drivers, subjects living near roads with intense traffic and suburban residents) and rural residents. Exposure was characterized by urinary excretion of S-phenylmercapturic acid (S-PMA), a biomarker of benzene exposure, and by ambient UFP. There were clear stepwise gradients with respect to ambient UFP, S-PMA excretion and oxidative DNA damage with rural subjects < suburban subjects < residents living near highly trafficed roads

CYP1A1 plays an important role in the metabolic activation of polycyclic aromatic hydrocarbons (PAH), carcinogenic components of air pollution. The influence of CYP1A1 genotype (*2A, *2B and *4) on the levels of lymphocyte bulky DNA adducts and the frequency of cells with aberrant chromosomes was assessed in 194 non-smoking subjects in whom recent exposure to environmental tobacco smoke (ETS) and airborne particulate-associated PAH were measured during two consecutive seasons (winter and summer). While CYP1A1*4 had no consistent effect on either biomarker of genetic damage, the levels of both biomarkers responded in a parallel fashion to changes in exposure/CYP1A1*2A genotype combinations during both seasons. Specifically, the levels of both biomarkers were increased in carriers of at least one CYP1A1*2A allele, as compared with CYP1A1*1 homozygotes, in subjects with ETS exposures >0.8 h/day during the previous 4 days and mean personal exposure to benzo[a]pyrene <0.9 ng/m3 during the previous 24 h (all P < 0.05). Outside these exposure limits the differential effect in CYP1A1*2A variants was lost. Although the numbers of subjects with the CYP1A1*2B polymorphism was small, the same trend appeared to be followed in this case. These effects are interpreted as resulting from differential induction of CYP1A1 expression in CYP1A1*2A and CYP1A1*2A/*2B carriers by components of ETS-polluted air at levels of exposure readily suffered by large segments of the general population and suggest that subjects with these genotypes may have increased susceptibility to the genotoxic effects of ETS.

Exposure to diesel exhaust particles (DEP) via the gastrointestinal route may impose risk of cancer in the colon and liver. We investigated the effects of DEP given in the diet to Big Blue rats by quantifying a panel of markers of DNA damage and repair, mutation, oxidative damage to proteins and lipids, and antioxidative defence mechanisms in colon mucosa cells, liver tissue and the blood compartment. Seven groups of rats were fed a diet with 0, 0.2, 0.8, 2, 8, 20 or 80 mg DEP/kg feed for 21 days. DEP induced a significant increase in DNA strand breaks in colon and liver. There was no effect on oxidative DNA damage (8-oxodG) in colon or liver DNA or in the urine. However, the mRNA expression of OGG1, encoding an enzyme involved in repair of 8-oxodG, was increased by DEP in both liver and colon. DNA adduct levels measured by 32P-post-labelling were elevated in colon and liver, and the expression of ERCC1 gene was affected in liver, but not in colon. In addition to these effects, DEP exposure induced apoptosis in liver. There was no significant change in mutation frequency in colon or liver. The levels of oxidative protein modifications (oxidized arginine and proline residues) were increased in liver accompanied by enhanced vitamin C levels. In plasma, we found no significant effects on oxidative damage to proteins and lipids, antioxidant enzymes or vitamin C levels. Our data indicate that gastrointestinal exposure to DEP induces DNA adducts and oxidative stress resulting in DNA strand breaks, enhanced repair capacity of oxidative base damage, apoptosis and protein oxidation in colon mucosa cells and liver.

The contribution of oxidative stress, different types of DNA damage and expression of DNA repair enzymes in colon and liver mutagenesis induced by 2-amino-3-methylimidazo [4,5-f]quinoline (IQ) was investigated in four groups of six Big Blue rats fed diets with 0, 20, 70, and 200 mg IQ/kg for 3 weeks. There were dose-response relationships of DNA adducts ((32)P-postlabeling) and DNA strand breaks (comet assay) in colon and liver tissues, with the highest levels of DNA adducts and strand breaks in the colon. There was dose-dependent induction of mutations in both the colon and the liver, and the same IQ dose produced two-fold more cII mutations in the liver compared with the colon. The IQ-induced mutation spectrum in the colon was not significantly different to that of control rats. The expression of ERCC1 and OGG1 was higher in the colon than liver, and was unaffected by the IQ diet. Investigations of oxidative stress biomarkers produced inconclusive results. Oxidative DNA damage detected by the endonuclease III enzyme and 7-hydro-8-oxo-2'-deoxyguanosine in colon, liver and/or urine was unaltered by IQ. However, there was increased level of gamma-glutamyl semialdehyde in liver proteins, indicating a higher rate of protein oxidation in the liver following IQ administration. In plasma and erythrocytes there were unaltered levels of oxidized protein, malondialdehyde, and antioxidant enzyme activities (superoxide dismutase, glutathione peroxidase, catalase, glutathione reductase) indicating no systemic oxidative stress. However, the level of total vitamin C was increased in plasma, with the largest fraction being in the reduced form. In conclusion, our results indicate that DNA adducts rather than oxidative stress are responsible for the initiation of IQ-induced carcinogenesis of the liver and colon. A lower frequency of mutations in the colon than in the liver could be related to higher expression of DNA repair enzymes in the former.

A host cell reactivation (HCR) assay was employed to study the capacity of a normal and three repair-deficient human lymphoblastoid cell lines to repair DNA damage induced by UV irradiation and the aromatic amines 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and N-acetyl-2-aminofluorene (AAF) respectively. The cell line belonging to xeroderma pigmentosum complementation group C (XP-C) removed all three types of damage less efficiently than the normal cell line, but more efficiently than the cell line belonging to xeroderma pigmentosum complementation group D (XP-D). The cell line belonging to complementation group B of Cockayne's syndrome (CS-B) showed reduced host cell reactivation. Fibroblasts from CS-B patients have reduced gene-specific DNA repair, but normal total genomic DNA repair, thus our data suggest that the HCR assay measures the capacity for gene-specific DNA repair. In the XP-D cell line, which had practically no DNA repair capacity, AAF adducts had a more potent inhibitory effect on gene expression than UV and PhIP adducts. When corrected for this inhibitory effect, the wild-type, XP-C and CS-B cell lines repaired low levels of AAF and UV adducts with similar efficiencies, however, PhIP adducts were repaired less efficiently.

Transplacental transfer of genotoxic material has been determined by measuring the polycyclic aromatic hydrocarbon-albumin adduct level in serum isolated from the mother and the umbilical cord using a competitive ELISA assay and the antibody (8E11) against benzo[a]pyrene (B[a]P) tetrols. Smoking women (median 5.54 fmol B[a]P equiv/microgram albumin; 21 cases) and non-smoking women living in rural areas (median 4.99; 30) had higher adduct levels than non-smoking women living in suburbia (median 4.09; 37), whereas non-smoking women living in the city of Aarhus had an intermediate level (median 4.82; 40). Exposure to passive smoking did not modify the adduct levels. When all non-smoking cases were combined, the transport time to/from the home became a major contributing factor to the adduct level. The median adduct level in umbilical cord blood was significantly lower than in maternal blood, the maternal/fetal ratio being approximately 1.3, and a positive association between the adduct levels in the mother and umbilical cord blood was observed. The frequency of the GSTM1 null genotype in the study population, females aged 19-44, was 55.4%, but the GSTM1 genotype did not significantly alter the serum albumin adduct level. This study indicates that the competitive ELISA to detect B[a]P bound to serum albumin is sensitive enough to detect differences in the burden of genotoxic compounds in non-occupational exposed individuals. The lower adduct level in people living in suburbia suggests that local production of incomplete combustion products, like vehicle exhaust or heat generation, is the major contributing factor to genotoxic compounds in the general environment.

It is known that consumption of cruciferous vegetables protects against the chemical induction of cancer in many organs. It has been suggested that this protection is mediated through an effect on the cytochrome P450 monooxygenase system. This system is responsible for the activation of a number of chemical carcinogens to their ultimate forms. In the present study, the effect of indole-3-carbinol (I3C) and 5,6-benzoflavone (5,6BF) on the expression of cytochrome P450IA1 in rat colon and liver has been investigated. Cytochrome P450IA1 mRNA was induced in colon following a single oral administration of I3C or 5,6BF. A biphasic induction profile was obtained with maxima at 4 and 16 h post-administration. Both inducers caused an approximately 2-fold increase in P450IA1 mRNA at 4 h and a 10-fold increase at 16 h. In contrast, both cytochrome P450IA1 and IA2 mRNAs was increased over the control between 4 and 24 h. The total amount of P450IA mRNAs in liver at 4 and 16 h was increased about 2- and 4-fold respectively by I3C; 5,6BF induced the P450IA mRNAs 4- and 5-fold respectively. The expression of cytochrome P450IA1 and IA2 is induced by I3C and several flavones present in cruciferous vegetables. This suggests that one of the protective effects of cruciferous vegetables in the reduction of chemically induced cancer may be regulation of cytochrome P450s involved in the metabolism of the chemical carcinogens.

A large number of chemical carcinogens require metabolic activation before they are biologically active. The metabolism of a few of these compounds has been investigated in cultured human tissues and cells and the metabolism has been compared with the metabolism in organs from experimental animals in which the compounds induce cancer. Generally, only quantitative differences could be observed between animal and human tissues. The development of new methods to detect carcinogen-DNA adducts makes it feasible to study and compare the metabolism in human and animal tissues of an increasing number of potential human carcinogens. Furthermore, construction of cell lines, expressing the human forms of the carcinogen metabolizing enzymes, by biotechnology provides a new model to study the metabolism and to monitor for genetic markers in the same cells.

The role of prostaglandin H synthase (PHS) in the metabolism of 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (BP-7,8-diol) has been examined in short-term explant cultures of hamster and human tracheobronchial tissues. Labeled BP-7,8-diol was incubated with the explants in the presence and absence of the PHS substrate arachidonic acid (20:4) and the PHS inhibitor indomethacin. The addition of 10 microM to 200 microM 20:4 to incubations of hamster trachea with 5 microM BP-7,8-diol caused significant increases in the formation of 7r,8t-dihydroxy-9t,10t-epoxy-7,8,9,10-tetrahydrobenzo[ a]pyrene (anti-BPDE). These increases were not seen when 1 microM or 20 microM BP-7,8-diol was employed. The stimulation of anti-BPDE formation was observed after incubations of from 1 to 48 h. This stimulation was inhibited to the basal level by 20 microM indomethacin, supporting the role of PHS in the response. No effect of 20:4 was seen on the uptake of BP-7,8-diol by the tracheas or on the formation of water-soluble metabolites. Significant increases in covalent binding of BP-7,8-diol metabolites to DNA of the tracheal epithelium were also elicited by the addition of 20:4, however these increases were not well correlated quantitatively with the increases in anti-BPDE formation. H.p.l.c. profiles of deoxynucleoside adducts from basal and 20:4-stimulated incubations were qualitatively identical. Far greater variability of metabolism was seen in human bronchus explants, but 20:4-dependent increases in anti-BPDE formation could be demonstrated in those tissues as well. Inhibition of this stimulation by indomethacin was either absent or incomplete. This variation in the effect of indomethacin was explained by the examination of the products of 20:4 metabolism by the two tissues. Hamster trachea produced almost exclusively PHS metabolites whereas human bronchus yielded predominantly products of lipoxygenases, enzymes insensitive to indomethacin. In conclusion, this study indicates that co-oxygenation of chemical carcinogens can occur in hamster and human tracheobronchial tissues. The concentration-dependence observed with BP-7,8-diol, however, suggests that this pathway is of minor importance in the activation of BP in these tissues.

The metabolism of benzo[a]pyrene, aflatoxin B1, N-nitrosodimethylamine, N-nitrosoethylmethylamine, and N-nitrosopyrrolidine has been studied in cultures of normal human and rat urinary bladder epithelial cells. The cultures were incubated with radioactively labeled carcinogens for 24 h, and the metabolism was assayed by binding of reactive metabolites to DNa and by the release of metabolites into the medium. Only slight variation in binding level of benzo[a]pyrene to DNa among the three human bladder cell lines was seen, the level of binding being higher than to rat DNA. The major benzo[a]pyrene-DNA adduct (80%) in human bladder cells eluted prior to the adducts formed by reaction of 7,8-dihydroxy-9,10-epoxy - 7,8,9,10-tetrahydrobenz[a]pyrene with guanine by high pressure liquid chromatography, but has yet to be identified. The benzo[a]pyrene-DNA adducts were quickly removed and only about 10% of the radioactivity remained associated with human bladder DNA 72 h post-treatment with benzo[a]pyrene. The 7,8- and 9,10-diols of benzo[a]pyrene were the major organo-soluble metabolites formed by both rat and human bladder cells. The primary benzo[a]pyrene metabolites were conjugated to a minor extent only. The highest level of modification of DNA was seen in the case of N-nitrosodimethylamine. N-Nitrosopyrrolidine was oxidized in both the alpha-and beta-position by all three cell lines, the oxidation at the alpha-position being predominant. No binding to DNA was detectable with N-nitrosoethylmethylamine, although this compound was metabolized as measured by the formation of CO2 and aldehydes. These results add the urinary bladder to the list of human organs which have been shown to metabolize chemical carcinogens into electropositive metabolites. However, qualitative differences exist between the data from bladder cells and those from other human organs.

The effect of a single treatment with the skin tumor-promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) on the expression of the cellular proto-oncogenes, c-myc, c-rasHa, c-rasKi and c-fos was examined in the non-tumorigenic human bladder epithelial cell line HCV 29. TPA (1 microgram/ml) increased the transcription of the c-fos gene of HCV 29 at least 50-fold, and this stimulation was observed within minutes. The response was transient, and was accompanied by a rapid and transient change in cell morphology. The expression of c-myc, c-rasHa and c-rasKi were not enhanced by the TPA treatment. These results show that human bladder epithelial cells respond to a known skin tumor-promoter, TPA, by altering the transcription of a specific proto-oncogene in these cells.

Primary cultures of epithelial and fibroblast cells derived from human oral mucosa were studied for the ability to activate a tobacco smoke carcinogen, benzo[a]pyrene (BP). The cells were exposed to benzo[a]pyrene for 18 h. The cell-free medium was extracted with ethylacetate/acetone, and high-pressure liquid chromatography analysis of this fraction revealed that BP tetrols and diols were the major metabolites formed by both epithelial and fibroblast cells. However, the epithelial cells had a much higher rate of biotransformation of BP as measured by binding to cellular DNA. The mean binding level to human buccal mucosal DNA was among the highest observed in stratified human epithelia. The major BP-DNA adduct was formed by the reaction of the 'bay-region' BP diolepoxide with the exocyclic 2-amino group in guanine. In contrast to human cells, BP phenols and BP 9,10-diol were the major metabolites produced by primary epithelial and fibroblast cells derived from rat buccal mucosa. The DNA binding levels of BP in the two rat cell types were identical, and the binding level was several-fold lower than in the human epithelial cells. When an established rat tongue epithelial cell line (RTE 2) was treated with polycyclic aromatic hydrocarbons--BP and 7,12-dimethylbenz[a]-anthracene--a slight toxic effect was observed. Our results indicate that primary cultures of oral mucosa are able to metabolize BP into its ultimate carcinogenic form at a rate similar to or higher than other potential target tissues for BP-induced carcinogenesis.

The overall metabolism of 1,2-dimethylhydrazine, and organotropic colon carcinogen in rodents, has been studied using human colon explant cultures. The binding level of 1,2-dimethylhydrazine to DNA which in this study includes both reaction of metabolites with DNA and incorporation of radioactive metabolites into DNA, showed a 100-fold variation among the 120 people studied. When different anatomical colonic sites were compared, the highest mean binding levels were found in the ascending and sigmoid colon. No significant difference in the median and mean binding levels were observed in nontumorous colon obtained surgically from patients with colon cancer and colon obtained from immediate autopsy, but decreased mean binding levels were seen in tissues obtained by surgery from patients with non-cancerous colonic disorders. Several exogenous chemicals were found to modify the metabolism. When the colon explants were co-incubated with 1,2-dimethylhydrazine and these chemicals, the binding level of 1,2-dimethylhydrazine to DNA was (a) increased by either indole 3-carbinol or phenobarbital, (b) decreased with disulfiram, butylated hydroxytoluene, or taurodeoxycholic acid, and (c) unaltered by lithocholic acid.

Food samples collected in Murang'a district, Kenya are known to be contaminated with a mycotoxin, aflatoxin B1 (AFB), and a positive correlation exists between the dietary intake of AFB and the incidence of liver cancer. When urine samples collected in this district were analyzed for the presence of 2,3-dihydro-2-(7'-guanyl)-3-hydroxyaflatoxin B1 (AFB-GuaI) by h.p.l.c., 6 of 81 samples had a detectable level of a compound whose fluorescence spectrum was identical to chemically synthesized AFB-GuaI as confirmed by photoncounting fluorescence spectrophotometry. These results are an indication of interaction between the ultimate carcinogenic form of AFB and cellular nucleic acids in vivo and further support the hypothesis that AFB may play an important role in the etiology of human liver cancer.