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Theoretically, both synthetic endocrine disrupting chemicals (S-EDCs) and natural (exogenous and endogenous) endocrine disrupting chemicals (N-EDCs) can interact with endocrine receptors and disturb hormonal balance. However, compared to endogenous hormones, S-EDCs are only weak partial agonists with receptor affinities several orders of magnitude lower. Thus, to elicit observable effects, S-EDCs require considerably higher concentrations to attain sufficient receptor occupancy or to displace natural hormones and other endogenous ligands. Significant exposures to exogenous N-EDCs may result from ingestion of foods such as soy-based diets, green tea and sweet mustard. While their potencies are lower as compared to natural endogenous hormones, they usually are considerably more potent than S-EDCs. Effects of exogenous N-EDCs on the endocrine system were observed at high dietary intakes. A causal relation between their mechanism of action and these effects is established and biologically plausible. In contrast, the assumption that the much lower human exposures to S-EDCs may induce observable endocrine effects is not plausible. Hence, it is not surprising that epidemiological studies searching for an association between S-EDC exposure and health effects have failed. Regarding testing for potential endocrine effects, a scientifically justified screen should use in vitro tests to compare potencies of S-EDCs with those of reference N-EDCs. When the potency of the S-EDC is similar or smaller than that of the N-EDC, further testing in laboratory animals and regulatory consequences are not warranted.

Health impacts of inhalation exposure to engineered nanomaterials have attracted increasing attention. In this paper, integrated analytical techniques with high sensitivity were used to study the brain translocation and potential impairment induced by intranasally instilled copper nanoparticles (CuNPs). Mice were exposed to CuNPs in three doses (1, 10, 40 mg/kg bw). The body weight of mice decreased significantly in the 10 and 40 mg/kg group (p

Combustion of biomass and wood for residential heating and/or cooking contributes substantially to both ambient air and indoor levels of particulate matter (PM). Toxicological characterization of ambient air PM, especially related to traffic, is well advanced, whereas the toxicology of wood smoke PM (WSPM) is poorly assessed. We assessed a wide spectrum of toxicity end points in human A549 lung epithelial and THP-1 monocytic cell lines comparing WSPM from high or low oxygen combustion and ambient PM collected in a village with many operating wood stoves and from a rural background area. In both cell types, all extensively characterized PM samples (1.25-100 mu g/mL) induced dose-dependent formation of reactive oxygen species and DNA damage in terms of strand breaks and formamidopyrimidine DNA glycosylase sites assessed by the comet assay with WSPM being most potent. The WSPM contained more polycyclic aromatic hydrocarbons (PAH), less soluble metals, and expectedly also had a smaller particle size than PM collected from ambient air. All four types of PM combined increased the levels of 8-oxo-7,8-dihydro-2'-deoxyguanosine dose-dependently in A549 cells, whereas there was no change in the levels of etheno-adducts or bulky DNA adducts. Furthermore, mRNA expression of the proinflammatory genes monocyte chemoattractant protein-1, interleukin-8, and tumor necrosis factor-alpha as well as the oxidative stress gene heme oxygenase-1 was upregulated in the THP-1 cells especially by WSPM and ambient PM sampled from the wood stove area. Expression of oxoguanine glycosylase 1, lymphocyte function-associated antigen-1, and interleukin-6 did not change. We conclude that WSPM has small particle size, high level of PAR, low level of water-soluble metals, and produces high levels of free radicals, DNA damage as well as inflammatory and oxidative stress response gene expression in cultured human cells.

published

The toxicity of a range of inorganic (Ag, Cu, Ni, Al(2)O(3), SiO(2), TiO(2) and ZrO(2)) nanoparticles (NP) and their corresponding metal salt or bulk metal oxide were screened for toxicity toward the earthworm Eisenia fetida using the limit-test design (1000 mg/kg). This study provides the first ecotoxicological life history trait data on earthworms for each these NPs, as well as for AgNO(3), Al(2)O(3), SiO(2), TiO(2) and ZrO(2). Significant effects were observed on survival for AgNO(3) (2.5% of controls), CuCl(2) (17.5% of controls) and NiCl(2) (32.5% of controls) and on reproduction (AgNO(3), CuCl(2), NiCl(2), Ag-NP, Cu-NP, TiO(2)-NP); with total reproductive failure in both silver treatments. Ag-NP, Cu-NP and TiO(2)-NP were the only NPs that caused toxic effects to E. fetida. The toxicity could not be singularly related to particle size or zeta potential or to the inherent element constituting the NPs (e.g. Ag).

The objective of the present study was to investigate the toxicity of silver nanoparticles (Ag NPs) in vitro . Silver ions (Ag+) have been used in medical treatments for decades whereas Ag NPs have been used in a variety of consumer products within recent years. This study was undertaken to compare the effect of well characterized, PVP-coated Ag NPs (69 nm ± 3 nm) and Ag+ in a human monocytic cell line (THP-1). Characterization of the Ag NPs was conducted in both stock suspension and cell media with or without serum and antibiotics. By using the flowcytometric annexin V/propidium iodide (PI) assay, both Ag NPs and Ag+ were shown to induce apoptosis and necrosis in THP-1 cells depending on dose and exposure time. Furthermore, the presence of apoptosis could be confirmed by the TUNEL method. A number of studies have implicated the production of reactive oxygen species (ROS) in cytotoxicity mediated by NPs. We used the fluorogenic probe, 2′,7′-dichlorofluorescein to assess the levels of intracellular ROS during exposure to Ag NPs and Ag+. A drastic increase in ROS levels could be detected after 6–24 h suggesting that oxidative stress is an important mediator of cytotoxicity caused by Ag NPs and Ag+.

Little is known about the impact of genetic variation on the genetic damage induced by urban air pollution or environmental tobacco smoke (ETS) in exposed populations. The levels of bulky DNA adducts ( 32P-postlabelling, nuclease P1 enrichment) and chromosomal aberrations were measured in lymphocytes of 194 non-smoking students living in the city of Athens, and the rural region of Halkida, Greece. In these individuals personal exposure to PAH was also measured. Furthermore, genetic polymorphisms were examined in cytochromes P450 1A1, 1B1, in the GSTM1, GSTP1 and GSTT1 as well as in microsomal epoxy hydrolase (EPHX) genes. Subjects with the CYP1*2A mutant genotype also suffering significant ETS exposure tended to exhibit higher adduct levels and % aberrant cells. In contrast, CYP1B1 polymorphisms seemed to have an impact on the DNA adduct levels only among individual with negligible ETS exposure. Subjects carrying both the CYP1*2A mutant genotype and the GSTM1 null genotype tended to have higher DNA adduct levels. A similar effect was also observed with the combined CYP1A1*2A/GSTP1 (Ile/Val) and the CYP1A1*2A/mEH "slow" polymorphisms. In both cases, the effect was more pronounced among subjects with higher levels of ETS exposure. Stepwise restriction of the observations to subjects characterised by (a) GSTP1 mutant, (b) GSTM1 null, (c) mEH "slow" (His139His) genotypes and (d) ETS exposure resulted in a significant trend of increasing DNA adduct levels only among individuals with at least one CYP1A1*2A mutated allele, illustrating the importance and complexity of gene-exposure and gene-gene interactions in determining the level of genotoxic damage on an individual levels.

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.

Exposure to PCBs may be an etiologic factor for breast cancer. The cytochrome P450 1B1 (CYP1B1) and catechol-O-methyltransferase (COMT) enzymes are involved in estrogen metabolism and PCB metabolism, both of which may relate to breast cancer susceptibility. Polymorphisms in genes regulating these enzymes control efficiency. Our objective was to assess whether CYP1B1 and COMT gene polymorphisms modulate the effect of PCBs in breast cancer risk, among postmenopausal Danish women. Neither CYP1B1 Leu432Val polymorphisms nor adipose tissue PCBs were independently associated with breast cancer risk. When assessing the independent effect of the COMT Val158Met polymorphism, we observed reduced risk for breast cancer amongst hormone replacement therapy using women who were homozygous carriers of the variant allele compared with those carrying the wild-type variant (RR = 0.41; 95% CI: 0.29-0.89). We found no statistically significant interactions between any of the PCB groups and CYP1B1 or COMT polymorphisms on the risk of breast cancer.

The levels of bulky DNA adducts were measured by (32)P-post-labelling in lymphocytes of 194 non-smoking students living in the city of Athens and the region of Halkida, Greece, once in the winter and again in the following summer. Personal exposures to particulate-bound polycyclic aromatic hydrocarbons (PAH) were significantly higher in Athens subjects during both seasons. There was hardly any diagonal radioactive zone in the pattern of DNA adducts observed. Highest adduct levels were observed in a sub-group of subjects living in or near the Halkida Institute campus, which was located in rural surroundings with a minimal burden of urban air pollution. The remaining Halkida subjects had intermediate levels, while Athens subjects showed the lowest levels. This trend, which was observed over both monitoring seasons, consistently paralleled the variation in three markers of exposure to environmental tobacco smoke (ETS), namely (i) declared times of exposure to ETS during the 24 h prior to blood donation, (ii) plasma cotinine levels and (iii) chrysene/benzo[g,h,i]perylene ratios in the profile of personal PAH exposure. Furthermore, among the Halkida campus area subjects (but not the remaining subjects) positive correlations were observed between DNA adducts and (i) measured personal exposures to chrysene or benzo[a]pyrene, (ii) time of declared ETS exposure and (iii) chrysene/benzo[g,h,i] perylene ratios. These correlations suggest that, for a group suffering minimal exposure to urban air pollution, exposure to ETS was a significant determinant of the observed DNA damage. Gender had a consistent and significant effect on adduct levels (males having higher levels), which remained significant even after multiple regression analysis. Habitual consumption of roasted meat was significantly associated with an enhancement of adduct levels and the effect was strengthened when only individuals unexposed to ETS were taken into consideration. No significant effects were observed for other dietary parameters or factors reflecting exposure to air pollution.

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.

Much of the concerns regarding engineered nanoparticle (NP) toxicity are based on knowledge from previous studies on particles in ambient air or occupational situations. E.g., the effects of exposure to silica dust particles have been studied intensely due to the carcinogenicity of crystalline silica. However, the increasing usage of engineered amorphous silica NPs has emphasized the need for further mechanistic insight to predict the consequences of exposure to the amorphous type of silica NPs. The present study focused on the in vitro biological effects following exposure to well-dispersed, BSA-stabilized, amorphous silica NPs whereas unmodified silica NPs where included for reasons of comparison. The cytotoxicity of the silica NPs was investigated in six different cell lines (A549, THP-1, CaCo-2, ASB-XIV, J-774A.1, and Colon-26) selected to explore the significance of organ and species sensitivity in vitro. Viability data demonstrated that macrophages were most sensitive to silica NP and interestingly, murine cell lines were generally found to be more sensitive than comparable human cell lines. Further studies were conducted in the human epithelial lung cell line, A549, to explore the molecular mechanism of silica toxicity. Generation of reactive oxygen species, one of the proposed toxicological mechanisms of NPs, was investigated in A549 cells by the dichlorofluorescin (DCF) assay to be significantly induced at NP concentrations above 113 μg/mL. However, induction of oxidative stress related pathways was not found after silica NP exposure for 24 h in gene array studies conducted in A549 cells at a relatively low NP concentration (EC20). Up-regulated genes (more than 2-fold) were primarily related to lipid metabolism and biosynthesis whereas down-regulated genes included several processes such as transcription, cell junction, extra cellular matrix (ECM)-receptor interaction and others. Thus, gene expression data proposes that several cellular processes other than oxidative stress could be affected by exposure to silica NPs.

Cytochrome CYP1A1 gene expression, induced by polycyclic aromatic hydrocarbons and dioxins, eg. 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), is regulated mainly at the level of transcription. Inducible activation of the CYP1A1 promotor is mediated by a ligand-dependent transcription factor dimer complex including the aryl hydrocarbon receptor (AHR) and the AHR nuclear translocator (ARNT) proteins. Additional factors seem to be involved in tissue- and cell-specific modification of the induction process. In the present study HepG2 and MCF-7 cell lines were used to examine a possible cell-specific autoregulation of CYP1A1 promotor function. Chimeric CYP1A1-CAT reporter constructs and a human CYP1A1 cDNA expression plasmid were used in transient co-expression experiments. In HepG2 cells co-expression of increasing amounts of CYP1A1 cDNA significantly down-regulated constitutive as well as the TCDD-induced CYP1A1 promotor driven CAT activity. In contrast, co-transfection of MCF-7 cells with a 3-fold molar excess of CYP1A1 cDNA relative to the CYP1A1-CAT reporter construct caused an approximately 2-fold increase in the TCDD-induced CAT activity, whereas no effect was observed on constitutive promotor activity. This autoregulatory mechanism(s) of the human CYP1A1 gene product was independent of specific 5' flanking promotor segments tested. RT-PCR analyses did not indicate any changes in mRNA level of AHR and ARNT in the co-transfection studies. Thus these studies show that the human CYP1A1 gene is exposed to cell-specific autoregulation, probably achieved via different functions of trans-acting factors.

The lymphocyte bulky PAH-DNA adduct levels have been studied in persons occupationally exposed to ambient air pollution. The exposure group consisted of 90 healthy, nonsmoking bus drivers from the Copenhagen area, divided into three exposure groups according to driving area, and 60 rural controls (smokers and non-smokers). PAH-DNA adducts were determined by 32P-postlabelling with the butanol enrichment procedure. The bus drivers answered a comprehensive questionnaire on passive smoking, residential area, diet and other potential confounding variables. A significantly higher adduct level was observed in bus drivers working in central Copenhagen (1.214 fmol/microg DNA, n = 49) compared with both those driving in the dormitory (median: 0.507 fmol/microg DNA, P = 0.046, n = 16) and suburban (median: 0.585 fmol/microg DNA, P = 0.041, n = 25) areas. All three groups had higher adduct levels than rural controls (0.074 fmol/microg DNA, n = 60, P < 0.001). No significant influence on adduct levels was demonstrated from potential confounders, including smoking and diet. The effect of the metabolizing enzymes, GSTM1 and NAT2, on adduct levels was investigated. No statistically significant effects were observed on adduct levels from GSTM1 or NAT2, either individually or combined, but a non-significant trend was seen for individuals with GSTM1*0/0 (null), since they had higher adduct levels in all exposure groups. This study demonstrated that lymphocyte PAH-DNA adduct levels were related to levels of exposure to urban air pollution and indicated that these adducts might be helpful as a means of classifying better different exposure groups for epidemiological studies. Furthermore, it demonstrated the ability of 32P-postlabelling to discern small differences in low exposure to ambient air pollution and suggested a possible effect of GSTM1*0/0 on DNA adduct levels.

Inhibition of gap junctional intercellular communication (GJIC) is a predictive short term test for tumor promoting activity. A new metabolic cooperation assay has been developed, which takes the cytochrome P-450 metabolism into account. In this assay the inhibitory activity of tobacco smoke condensates (CSC) and CSC fractions from high and low tar cigarettes was tested. CSC of both high and low tar cigarettes and fractions thereof contained tumor promoting activity. The tar yield of the cigarettes did not closely reflect the effects in the GJIC assay and the major constituent nicotine had no effect. The effect was only marginally greater in cells expressing different cytochrome P-450 enzymes, indicating that the active substances are not metabolized by these enzymes. The activities of CSC fractions were considerably lower than the activities in the unfractionated CSC. This may indicate that compounds in the CSC act strongly synergistically. Furthermore, CSC and CSC fractions synergistically inhibit GJIC with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate, indicating different mechanisms of action.

Air pollution effect on humans represents a major public health problem. Exposure to genotoxic compounds in the ambient air is evaluated using different biomarkers. In the present study we assessed DNA-adducts levels in apparently healthy people living and working in the city of Cotonou (Benin) in which exposure to air pollutants such as benzene and polycyclic aromatic hydrocarbons (PAHs) mainly benzo(a)pyrene has been evidenced. Rural inhabitants were enrolled as control group. Taxi-motorbike drivers, street food vendors, and gasoline salesmen were recruited in Cotonou whereas suburban residents were recruited in Godomey, 12 km from Cotonou. We found that taxi-motorbike drivers, roadside residents, street vendors, taxi-motor-bike drivers and gasoline sellers had significantly higher levels of DNA-adducts than suburban and village inhabitants (P

The role of metabolism of tumour promoters on the inhibition of intercellular communication was investigated in a modified V79 metabolic cooperation system. V79 cells, which stably express different rat cytochrome P450 enzymes (CYP1A1, CYP1A2 or CYP2B1), were used in the metabolic cooperation assay. The inhibitory effect on intercellular communication of four compounds was changed in cells expressing cytochrome P450 enzymes, compared to cells without. The phorbol ester TPA and di(2-ethylhexyl)phthalate blocked intercellular communication in all the cell lines tested, but expression of CYP1A1 enzyme reduced the inhibitory activity in these cells. Diethylstilbestrol caused inhibition only with cells containing cytochrome P450 enzymes. In contrast, the benzene metabolite hydroquinone inhibited metabolic cooperation preferentially in cells without cytochrome P450 enzymes. The inhibition of metabolic cooperation by another benzene metabolite, phenol, was not affected by the cytochrome P450 enzymes. The inhibitory activity of several chemicals that have not been tested previously was analysed in the new metabolic cooperation assay. The inhibitory activity of none of these chemicals was affected by cytochrome P450-associated metabolism. 7-Octylindolactam V was as potent as TPA, whereas the related indolactam V was 100-fold less active. The carcinogenic aromatic amine 4-aminobiphenyl, but not its primary metabolite 4-hydroxyaminobiphenyl, inhibited metabolic cooperation. Other known carcinogens, ochratoxin A, aflatoxin B1 and 4-nitrobiphenyl, did not inhibit metabolic cooperation in either V79 cells expressing or cells not expressing cytochrome P450. We conclude that cytochrome P450-associated metabolism plays an important role in the inhibition of gap junctional intercellular communication of some tumour promoters. The modified metabolic cooperation assay presented here is valuable for detecting some inhibitory chemicals which have been 'false negative' in previous assays for gap junctional intercellular communication. The assay also discloses that cytochrome P450 metabolism alters intercellular communication by a mechanism other than metabolism of the exogenous inhibitor.

It has long been known that environmental factors play an important role in the induction of cancer, whereas the attributable fraction to genetic factors is less. However twin studies indicate that gene-environment interaction is of great importance. The focus is here on low penetrance genes, especially on genes involved in the biotransformation of environmental carcinogens, enzymes involved in the defense against oxidative stress, and repair of DNA damage induced by the environmental carcinogen. Early studies focused on the effect of SNPs in xenobiotica metabolizing enzymes, eg. Members of the cytochrome P450 and GST families of enzymes, but as the technology to detect genetic profiles has progressed, the studies moved from SNP to haplotypes and more recently genome wide analysis. However, most of the published studies is focused on single SNP, or combinations of SNP rather than on haplotypes. Focus of this presentation will be on breast cancer risk mostly based upon studies in the Danish part of the EPIC cohort. Only few environmental risk factors for the induction of breast cancer has been identified, e.g. alcohol consumption, cigarette smoking. Members of the GST familie of enzymes are normally involved in the detoxification of environmental carcinogens and chemotherapeutic agents by conjugation process using reduced glutathione as a cofactor. Furthermore, they can be involved in the detoxification of reactive oxygen species. The role of several family members, GSTM1, GSTT1, GSTP1, GSTA1 and GSTO1 in breast cancer oncogenesis has been investigated in the Danish Epic cohort involving 79729 women aged 50-64 years, and born in Denmark. The design was nested-case control. The polymorphisms in GSTM1 and T1 are deletion mutations, whereas the polymorphism in P1 is the coding sequence, and thus results in an enzyme with an altered substrate specificity. However, these polymorphism did not have an significant effect on breast cancer risk neither alone or in combination with dietary intake. Similarly no association was found between GSTA1 polymorphism and breast cancer risk, whereas the GSTO1 *B/*b was associated with an increased risk, an association that was strongest in estrogen receptor positive breast cancer. Alcohol is considered a risk factor for breast cancer, an effect that could either be mediated either by its ability to metabolic transformation to the carcinogen acetaldehyde, induction of reactive oxygen species or induction of hormone metabolizing enzymes. Alcohol dehydrogenase is involved in the oxidative formation of acetaldehyde. A genetic polymorphism in ALD1C (Arg272Gln) associated with high activity (Arg272) was found to be associated with a 14% increased risk per 10 g alcohol/day. Glutathione peroxidase 1 (GPX) is an antioxidant enzyme. A genetic polymorphism Pro198Leu has been detected, and a low activity was associated was linked to an increased breast risk Alcohol intake was correlated with increased alcohol intake only in carriers of the allele coding for Pro(198). During the preparation of meat at high temperature, food mutagens are formed. Some of these compounds have shown to induced breast cancer in experimental animals. Aromatic amines are metabolized by N-acetyltransferase to acetylated products. Several polymorphisms have been detected in NAT2, and people were grouped into low and fast metabolisers. An increased risk of breast cancer was observed in people with high intake of red meat, but an interaction was only observed in the intermediate/fast acetylators. A few studies have also addressed the role of these genetic polymorphisms on the prognosis of breast cancer. However, the data is generally inconsistent, but both GST and manganese superoxide dismutase polymorphisms has been associated with the prognosis of the disease. Genetic polymorphisms in genes coding for xenobiotica metabolizing enzymes only play a minor role in the induction of breast cancer, however in the present of a well-defined exposure an interaction between gene and environmental risk occurs.

Ingestion of broccoli or other cruciferous vegetables inhibits the induction of cancer by chemicals and modifies some cytochrome P-450 enzyme activities. The effect of dietary broccoli on the levels of P450IA and IIB mRNA and proteins in rat liver and colon has been studied. Rats were fed a ten percent broccoli diet for 7 days. The expression of the cytochrome P-450 forms was altered to a different extent in the liver and colon. The level of total P450IA mRNA in the liver was increased by the broccoli together with the P450IA1 and IA2 proteins. Colonic P450IA1 mRNA and protein were induced by the broccoli diet, whereas only P450IA2 protein and not mRNA was detectable in colon, but the protein level was unaffected by the broccoli diet. Liver P450IIB and IIE1 proteins were increased by the broccoli diet, whereas the level of P450IIB mRNAs was not affected. In contrast, the P450IIB mRNA levels were reduced but the protein levels were increased in colon and we suggest that a feedback mechanism caused the decrease of the P450IIB mRNAs levels. Because the ratio between activation and deactivation may be an important risk determinant, we conclude that the protective effect of the broccoli diet on chemically induced tumors in rodents may be caused by the broccoli-induced changes in P450IA and IIB associated enzyme activities.

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

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Traffic related urban air pollution is a major environmental health problem in many large cities. Children living in urban areas are exposed to benzene and other toxic pollutants simultaneously on a regular basis. Assessment of benzene exposure and oxidative DNA damage in schoolchildren in Bangkok compared with the rural schoolchildren was studied through the use of biomarkers. Benzene levels in ambient air at the roadside adjacent to Bangkok schools was 3.95-fold greater than that of rural school areas. Personal exposure to benzene in Bangkok schoolchildren was 3.04-fold higher than that in the rural schoolchildren. Blood benzene, urinary benzene and urinary muconic acid (MA) levels were significantly higher in the Bangkok schoolchildren. A significantly higher level of 8-hydroxy-2'-deoxyguanosine (8-OHdG) in leukocytes and in urine was found in Bangkok children than in the rural children. There was a significant correlation between individual benzene exposure level and blood benzene (rs=0.193, P

P-glycoprotein (Pgp) plays a part in the intestinal uptake of xenobiotics and has been associated with susceptibility to ulcerative colitis. The aim of this study was to examine Pgp activity in relation to age, gender, medical treatment (rifampicin or ketoconazole) and the multidrug resistance (MDR1) gene single nucleotide polymorphisms (SNPs) G2677T and C3435T using the model drug digoxin.Pgp activity was estimated from the pharmacokinetics of orally administered digoxin in blood samples from 32 healthy subjects. MDR1 gene expression in duodenal biopsies was monitored by real-time quantitative RT-PCR (RQ-PCR) and Western blot analyses. MDR1 SNPs were determined by PCR-restriction fragment length polymorphism (PCR-RFLP). The effect of medical treatment was tested by open, randomized, cross-over treatment with rifampicin and ketoconazole.Rifampicin treatment resulted in increased Pgp activity, duodenal MDR1 mRNA expression and Pgp detection compared with that in the control group (p0.05 for all), Pgp activity being associated with duodenal MDR1 mRNA level (p0.05). Individuals homozygous for the 3435 wild-type allele (CC) showed higher Pgp activity (p0.05), whereas SNP 2677 apparently did not affect Pgp activity. No variation in Pgp in relation to age or gender was found.Our data confirm that rifampicin increases Pgp activity, by increasing MDR1 mRNA and Pgp levels. Moreover, we found that the wild-type allele of the synonymous polymorphism of MDR1 position 3435 confers a higher Pgp activity. These data support other findings suggesting an effect of Pgp on treatment response and disease susceptibility.

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); P0.001). 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); P0.001 and 1.50 +/- 0.12 versus 0.43 +/- 0.05 ng/m(3); P0.001, 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; P0.001). Finally, a significantly higher level of DNA strand breaks and a significantly lower level of DNA repair capacity were observed in Bangkok schoolchildren (P0.001). 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.

Background Cytochrome P4501A2 (CYP1A2) and N-acetyltransferase 2 (NAT2) are key enzymes in the metabolism of caffeine. The polymorphism of these genes facilitates the detection of fast and slow metabolizers, and if caffeine is causally related to stillbirth, we expect slow metabolizers to have a higher risk of stillbirth at any given intake of caffeine. GluthationeS-transferase a1 (GSTA1) may also be active in the metabolism of caffeine as it conjugates glutathione to aromatic amines. Our study, therefore, included analyses of the association between GSTA1 and stillbirth. Methods A nested case non-case study among women who participated in the Danish National Birth Cohort: 142 cases of singleton stillbirths and 157 controls of singleton live births. Results Slow oxidizer status (CYP1A2), slow acetylator status (NAT2), and low activity of GSTA1 were not individually associated with the risk of stillbirth [odds ratio (OR) 5 1.06, 95% confidence interval (95% CI) 0.67–1.67, OR 5 0.95, 95% CI 0.60–1.51, and OR 5 1.42, 95% CI 0.88–2.28, respectively]. We did, however, observe that subjects with a combination of slow CYP1A2, slow NAT2, and low GSTA1 genes had almost a 2-fold risk of stillbirth compared with subjects with other combinations of genotypes. Conclusions We found no link between any single genotype and the risk of stillbirth. An association between a combination of genotypes and stillbirth was discovered. Caffeine may be causally related to stillbirth, but larger studies using Mendelian randomization are needed to verify this.

We have shown previously that a high sucrose intake increases the background level of somatic mutations and the level of bulky DNA adducts in the colon epithelium of rats. The mechanism may involve either glucose or fructose formed by hydrolysis of sucrose. Male Big Blue ® rats were fed 30% sucrose, glucose, fructose or potato starch as part of the diet. Mutation rates and bulky DNA adduct levels were determined in colon and liver. The concentration of short-chain fatty acids and pH were determined in caecum, C-peptide was determined in plasma, biomarkers for oxidative damage and proliferation were determined in colon, and a metabonomic analysis was performed in plasma and urine. The sugars increased the mutation rates in colon and the bulky adduct levels in colon and liver to a similar extent. All sugars decrease the caecal concentration of acetic acid and propionic acid. The metabonomic studies indicated disturbed amino acid metabolism and decrease in plasma and urinary acetate as a common feature for all sugars and confirmed triglyceridemic effects of fructose. In conclusion, the genotoxicity may be related to the altered chemical environment in the caecum and thereby also in the colon but we found no related changes in insulin resistance or oxidative stress.

Environmental carcinogens contained in air pollution, such as polycyclic aromatic hydrocarbons, aromatic amines or N-nitroso compounds, predominantly form DNA adducts but can also generate interstrand cross-links and reactive oxygen species. If unrepaired, such lesions increase the risk of somatic mutations and cancer. Our study investigated the relationships between 22 polymorphisms (and their haplotypes) in 16 DNA repair genes belonging to different repair pathways in 1094 controls and 567 cancer cases (bladder cancer, 131; lung cancer, 134; oral-pharyngeal cancer, 41; laryngeal cancer, 47; leukaemia, 179; death from emphysema and chronic obstructive pulmonary disease, 84). The design was a case-control study nested within a prospective investigation. Among the many comparisons, few polymorphisms were associated with the diseases at the univariate analysis: XRCC1-399 Gln/Gln variant homozygotes [odds ratios (OR) = 2.20, 95% confidence intervals (CI) = 1.16-4.17] and XRCC3-241 Met/Met homozygotes (OR = 0.51, 95% CI = 0.27-0.96) and leukaemia. The recessive model in the stepwise multivariate analysis revealed a possible protective effect of XRCC1-399Gln/Gln in lung cancer (OR = 0.22, 95% CI = 0.05-0.98), and confirmed an opposite effect (OR = 2.47, 95% CI = 1.02-6.02) in the leukaemia group. Our results also suggest that the XPD/ERCC1-GAT haplotype may modulate leukaemia (OR = 1.28, 95% CI = 1.02-1.61), bladder cancer (OR = 1.38, 95% CI = 1.06-1.79) and possibly other cancer risks. Further investigations of the combined effects of polymorphisms within these DNA repair genes, smoking and other risk factors may help to clarify the influence of genetic variation in the carcinogenic process. © 2006 Oxford University Press.

Objectives To investigate the association between environmental tobacco smoke, plasma cotinine concentration, and respiratory cancer or death. Design Nested case-control study within the European prospective investigation into cancer and nutrition (EPIC). Participants 303 020 people from the EPIC cohort (total 500 000) who had never smoked or who had stopped smoking for at least 10 years, 123 479 of whom provided information on exposure to environmental tobacco smoke. Cases were people who developed respiratory cancers or died from respiratory conditions. Controls were matched for sex, age (plus or minus 5 years), smoking status, country of recruitment, and time elapsed since recruitment. Main outcome measures Newly diagnosed cancer of lung, pharynx, and larynx; deaths from chronic obstructive pulmonary disease or emphysema. Plasma cotinine concentration was measured in 1574 people. Results Over seven years of follow up, 97 people had newly diagnosed lung cancer, 20 had upper respiratory cancers (pharynx, larynx), and 14 died from chronic obstructive pulmonary disease or emphysema. In the whole cohort exposure to environmental tobacco smoke was associated with increased risks (hazard ratio 1.30, 95% confidence interval 0.87 to 1.95, for all respiratory diseases; 1.34, 0.85 to 2.13, for lung cancer alone). Higher results were found in the nested case-control study (odds ratio 1.70, 1.02 to 2.82, for respiratory diseases; 1.76, 0.96 to 3.23, for lung cancer alone). Odds ratios were consistently higher in former smokers than in those who had never smoked; the association was limited to exposure related to work. Cotinine concentration was clearly associated with self reported exposure (3.30, 2.07 to 5.23, for detectable/non-detectable cotinine), but it was not associated with the risk of respiratory diseases or lung cancer. Frequent exposure to environmental tobacco smoke during childhood was associated with lung cancer in adulthood (hazard ratio 3.63, 1.19 to 11.11, for daily exposure for many hours). Conclusions This large prospective study, in which the smoking status was supported by cotinine measurements, confirms that environmental tobacco smoke is a risk factor for lung cancer and other respiratory diseases, particularly in ex-smokers.

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 Blues 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 OGGI, encoding an enzyme involved in repair of 8-oxodG, was increased by DEP in both liver and colon. DNA adduct levels measured by 32 P-post-labelling were elevated in colon and liver, and the expression of ERCCI 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.

Earlier studies have indicated that sucrose possesses either co-carcinogenic or tumor-promoter effects in colon carcinogenesis induced by genotoxic carcinogens. In this study we investigated the role of sucrose on diesel exhaust particle (DEP)-induced genotoxicity in the colonic mucosa and liver. Big Blue rats were fed with DEP (0.8 ppm in feed) and/or sucrose (3.45% or 6.85% w/w in feed) for 3 weeks. DEP increased both DNA strand breaks and DNA adducts in colon. Interestingly, sucrose also increased the level of bulky DNA adducts in colon. DEP and sucrose had no effect on DNA strand-breaks and DNA adducts in liver. DEP and sucrose treatment did not have any effect on mutation frequency in colon and liver. Oxidative DNA damage detected as 8-oxodG (8-oxo-7,8-dihydro-2'-deoxyguanosine) and endonuclease III or formamidopyrimidine DNA glycosylase sensitive sites was unaltered in colon and liver. The mRNA expression levels of the DNA repair enzymes N-methylpurine DNA glycosylase ( MPG), 8-oxoguanine DNA glycosylase ( OGG1) and ERCC1 (part of the nucleotide excision repair complex) measured by reverse transcription-polymerase chain reaction were increased in liver by DEP feeding. In colon, expression was unaffected by DEP or sucrose feeding. Among biomarkers of oxidative stress, including vitamin C, malondialdehyde and protein oxidations (gamma-glutamyl semialdehyde and 2-amino adipic semialdehyde) in plasma and liver, only malondialdehyde was increased in plasma by sucrose/DEP feeding. In conclusion, sucrose feeding did not increase DEP-induced DNA damage in colon or liver.

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.

The rapid development of nanotechnologies has been accompanied by an increased concern for the safety of nanomaterials and consequently the number of publications on nanotoxicology has increased rapidly. However, inadequate characterization of the investigated nanomaterials has obscured risk assessment. Today, nanosilver is used in more manufacturer-identified consumer products than any other nanomaterial. A major concern is the possibility that these materials possess novel physico-chemical properties compared to the bulk counterparts. Our in vitro data suggest that the toxicity of nanosilver is comparable to that of silver ions. From a mechanistic perspective, ROS generation and oxidative stress, demonstrated by the hierarchical oxidative stress paradigm, is a generally accepted model to explain the toxic effects of various types of nanoparticles. We have previously reported that both silver ions and silver nanoparticles induce ROS, apoptosis and necrosis in A549 and THP-1 cells. This increase was found to correlate with increased gene expression of stress-related genes (e.g. HO-1 and HSP70). Additionally, global gene arrays were conducted to compare between silver ions and nanosilver. Despite significant increases in ROS levels, bulky DNA adduct levels only increased at highly cytotoxic silver concentrations whereas comet assay analysis showed no genotoxicity. Although increasing evidence suggests that nanosilver toxicity primarily is due to silver ions, the nanoparticle might exert different toxicity due to alternative uptake and intracellular dissolution. In correspondence, phagozytosing cells such as macrophages appear especially sensitive to silver nanoparticles compared to e.g. epithelial cells. These findings indicate the importance of evaluating the toxic contribution of nanoparticle dissolution.

Formation of carcinogen-DNA adducts in rat oral epithelial cells after treatment with cigarette smoke condensate (CSC) or chewing tobacco in the presence of ethanol was investigated using the 32P-postlabeling procedure. Concomitant treatment of the cells with ethanol increased the relative adduct level over that found in cells treated with tobacco smoke condensate only. Treatment with chewing tobacco resulted in slightly higher adduct levels than in controls. Treatment of the cells with ethanol did not significantly increase the uptake of a polycyclic aromatic hydrocarbon, benzo[j]fluoranthene, however, high tar CSC alone or in combination with ethanol significantly increased the uptake of radiolabeled benzo[j]fluoranthene, suggesting that increased uptake of the carcinogens may be one of the synergistic mechanisms of alcohol in oral carcinogenesis.

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 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.

Increasing levels of nitrate (NO3-) in drinking water in Denmark is of concern due to the possibility of an associated increase in long-term exposure to endogeneously formed N-nitroso compounds. Using a duplicate portion technique in combination with a qualitative description of diet and other background variables, the total nitrate intake in a Danish rural population and the contribution of drinking water to the total nitrate exposure is estimated. People drinking nitrate-free water have an intake of 37 mg NO3- per day. At 47 mg NO3- per litre, the exposure is increased to 89 mg, about 60% of which originates from the water. At 84 mg NO3- per litre, the daily exposure is 123 mg, 70% of which originates from the drinking water. These crude comparisons between three groups of people are supplemented with quantitative modelling of nitrate exposure at individual level. Apart from drinking water, consumption of vegetables is a major source of nitrate in this population. Using nitrate in overnight urine samples to quantify exposure is less accurate than the duplicate dietary portion technique and tends, in the present study, to underestimate the contribution of water-derived nitrate to total nitrate intake.

Cultured fetal human stomach, esophagus and liver activated benzo[a]pyrene (BP), aflatoxin B1 (AFB) and certain N-nitrosamines into metabolites that bound to cellular DNA. When the 3 organs were compared the highest level of activity was observed in the stomach. The interindividual variation was 10-fold and the amount of carcinogen-DNA adducts did not correlate with the sex or age of the fetus. The reaction products between BP or AFB and cellular DNA were investigated in liver explants. The carcinogen-DNA adduct patterns were identical to those observed in adult human tissues; BPDEI-Gua being the major adduct formed by BP and 2,3-dihydro-2-(7′-guanyl)-3-hydroxy-AFB by AFB. The results indicate that fetal organs can metabolize those oncogenic compounds at an early stage of the development, and that the metabolic pathways and DNA adducts are quite similar to those in experimental animals in which the compounds are carcinogenic.

The induction of cytochrome P-450IA1 by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was studied in eight human breast tumour cell lines. The cells were treated with various concentrations of TCDD for 24 h, and total RNA was isolated. The level of P-450IA1 RNA induced by 1 nM TCDD followed the order: MCF-7 greater than T47-D greater than ZR-75-1 greater than 3909 greater than 3522. AL-1, BT-20 and CAMA-1 did not respond to TCDD at the concentrations used. Northern blot analysis revealed 2 bands at 2.7 and 2.0 Kb, respectively, with the larger band being 6-fold more intense. The ratio was not changed by the TCDD treatment. TCDD induction did not change the benzo[a]pyrene-7,8-diol (BP-7,8-diol) metabolite profile compared with control cells, when cells were incubated with [3H]BP-7,8-diol for 24 h following the treatment with TCDD. These results demonstrate that different breast tumour cell lines vary greatly with respect to the basal expression levels of P-450IA1 RNA and its inducibility by TCDD. Furthermore, TCDD treatment does not change the relative distribution of BP-7,8-diol metabolites.

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.

1. 1. The activity of microsomal drug-metabolizing enzymes—azo reductase, nitroreductase, p- hydroxylation , N-demethylation, O-demethylation, NADPH cytochrome c reductase and cytochrome P P-450—in the olive baboon are lower than in other animal species, e.g. mouse, rat, guinea-pig. 2. 2. The level is comparable with the level in man for N-demethylation, azo reductase, O-demethylation and NADPH cytochrome c reductase. 3. 3. The ratio of the activity of the p- hydroxylation as compared with cytochrome P-450 is very similar in baboon and man. 4. 4. Arylsulfatase is localized in the lysosomal fraction and beta-glucuronidase is present more in the lysosomal than in the microsomal fraction.

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.

Using a competitive enzyme immunoassay, one or more substances recognized by a monoclonal antibody against aflatoxin B 1 were detected in human urine samples collected in Denmark. The concentration of urinary aflatoxin-like substances was equivalent to 0.0–6.5 ng aflatoxin B 1 /mg creatinine. A truly competitive interaction in the immunoassay was found between aflatoxin-like substances and aflatoxin B 1 . Aflatoxin-like substances could be isolated in small quantities from urine by affinity chromatography. The quantity of urinary aflatoxin-like compounds in the samples collected showed a skewed normal distribution (80 individuals). In order to explain the seemingly high level of aflatoxin-like material in urine samples from people living in a cold temperate climate, the source of aflatoxin-like compounds was investigated. In a dietary restriction study, potential dietary factors leading to excretion of aflatoxin-like compounds were investigated. Our data indicate that the excretion of these compounds by healthy Danes depends mainly on the food ingested 24–48 hr before urine samples were collected. In particular, the excretion of aflatoxin-like substances was increased when diets include beer, dairy products or meat. A map of the epitope recognized by the antibody was constructed from the results of competition studies with several AFB 1 analogues. The epitope map was used to draw chemical structures representing the minimal requirements for antibody recognition. An on-line search was conducted among the 98.2 × 10 6 structures in the Chemical Abstracts and Registry Databases (STN, Columbus, OH) and provided strong evidence that only aflatoxins or aflatoxin derivatives are recognized by the antibody. The possible chemical structures of the aflatoxin-like substances are discussed.

The effect of various co- and anti-carcinogens of colon carcinogenesis on the metabolism of benzo(a)pyrene (BP) in cultured rat colon is reported. Rat colon enzymatically converted BP into metabolites which bind to cellular macromolecules i.e., DNA and protein. Activity of aryl hydrocarbon hydroxylase (AHH) activity and binding levels of BP to macromolecules were higher in the descending colon when compared to other segments. The major metabolites of BP, extractable with ethylacetate, were quinones, tetrols, 7,8-diol and a peak containing 9,10-dihydroxy-9,10-dihydrobenzo(a)pyrene and 7,8,9-trihydroxy-7,8-dihydrobenzo(a)pyrene. The binding levels of BP to DNA and protein in the explant was lowered by co-incubation with 7,8-benzoflavone (7,8-BF) (3.6 and 18.0 μM), a known inhibitor of AHH, and with disulfiram (100 μM), an anti-oxidant. The absence of vitamin A in the media also resulted in a lower level of BP binding to DNA and protein and in lower activity of AHH. Pretreatment with known inducers of AHH such as phenobarbital (PB) or benz(a)anthracene (BA), did not have any significant effect on the binding levels of BP to DNA or on the AHH activity. of the bile acids investigated only taurodeoxycholic acid significantly increased the binding level of BP to DNA.