Effects of pretreatment with benzo[a]pyrene on the stereochemical selectivity of metabolic activation of benzo[a]pyrene to DNA-binding metabolites in hamster embryo cell cultures

The proportions of individual benzo[a]pyrene (BaP)-DNA adducts present in rodent embryo cell cultures change with the length of time of exposure to BaP; the major alteration is an increase in the proportion of (+)-anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydroBaP (BaPDE)-deoxyguanosine (dG) adduct (Sebti et al., Cancer Res., 45 (1984) 1594-1600). To determine if this change in the BaP-DNA adducts could result from the induction of enzymes involved in oxidation of BaP, hamster embryo cell cultures were exposed to acetone or BaP for 24 h and then the medium was replaced with fresh medium containing [3H]BaP. After 5 h the BaP-pretreated cells had a 30% higher level of binding of BaP to DNA and formed a greater proportion of (+)-anti-BaPE-dG adduct than the acetone-pretreated control group. Cells pretreated for 24 h with BaP and then exposed to [3H]BaP and Actinomycin D for 5 h had a lower level of binding of BaP to DNA and a lower amount of (+)-anti-BaPDE-deoxyguanosine adduct than cells pretreated with acetone and exposed to [3H]BaP for 5 h. In contrast, pretreatment for 24 h with BaP plus Actinomycin D followed by a 5-h exposure to [3H]BaP resulted in a decrease in overall binding of BaP to DNA but had no effect on the amount of (+)-anti-BaPDE-deoxyguanosine adduct. Actinomycin D treatment had no significant effect on either the total amount of BaP metabolized, the formation of primary and water-soluble BaP metabolites, or cell viability, but reduced [3H]uridine incorporation into RNA by more than 65% at all times. These results suggest that induction of specific isozymes of cytochrome P-450 may be involved in the time-dependent increase in the proportion of (+)-anti-BaPDE-DNA adducts in BaP-treated cells. The state of induction of specific isozymes of cytochrome P-450 and the ability of the BaP dose applied to induce them may be major factors in determining the proportion of BaP metabolized to (+)-anti-BaPDE, the most carcinogenic stereoisomer of BaPDE.     

DNA adduct formation in northern pike (Esox lucius) exposed to a mixture of benzo[a]pyrene, benzo[k]fluoranthene and 7H-dibenzo[c, g]carbazole: time-course and dose-response studies

The time-course and dose dependent formation of DNA adducts in juvenile northern pike (Esox lucius) following a single exposure to a mixture of benzo[a]pyrene (BaP), benzo[k]fluoranthene (BkF) and 7H-dibenzo[c,g]carbazole (DBC) were investigated by use of the (32)P-postlabelling assay. A complex adduct pattern was detected in liver and intestine of exposed fish. For the time-course studies fish were exposed either by oral administration or by intraperitoneal (i.p.) injection. Following a single i.p. injection of the mixture (40micromole/kg body weight of each substance) significantly elevated DNA adduct levels were detected in the liver after 1 day. Adduct levels were higher in liver than in intestine, in which significant elevation were detected from day 3 to 12. Following exposure via food (80micromole/kg body weight of each substance), adduct levels were detected in both liver and intestine 1 day after exposure, and continued to increase until day 3 in liver and day 6 in intestine. Calculation of a binding index, which compensates for differences in dosage, resulted in much higher adduct formation (five times in liver and 22 times in intestine) following oral exposure. Pikes receiving single oral doses of 12.5, 50, 100 or 200micromole/kg body weight of each substance exhibited significantly higher adduct levels in both liver and intestine compared to controls. Hepatic adduct levels were also higher in fish given 100 and 200micromole/kg compared to 12.5micromole/kg. Results from this study show that DNA adducts are rapidly formed in juvenile northern pike following both i.p. injection and feeding of a mixture of BaP, BkF and DBC. A maximum level was reached within a few days, which then persisted at approximately the same level for at least 9-12 days. The results also shows that higher levels of adducts were obtained following oral administration compared to i.p. injection, particularly in the intestine.     

Xenobiotic metabolizing enzymes in spawning English sole (Parophrys vetulus) exposed to organic-solvent extracts of marine sediments from contaminated and reference areas

Female English sole (Parophrys vetulus) within 1-2 days of spawning were exposed by i.m. injection to organic-solvent extracts of marine sediments at the following doses: Eagle Harbor (EHSE, contaminated site)--6.8 mg aromatic hydrocarbons (AHs)/kg body wt; Duwamish Waterway (DSE, contaminated site)--0.52 mg AHs and 0.040 mg chlorinated hydrocarbons (CHs)/kg body wt; Hood Canal (HCSE, reference site)--0.00090 mg AHs/kg body wt. Hepatic aryl hydrocarbon hydroxylase (AHH) activity, measured at spawning, was induced 10-, 23-and 2-fold by EHSE, DSE and HCSE, respectively, compared to sham and vehicle controls. Hepatic glutathione-S-transferase and epoxide hydrolase activities were not affected by any treatment. EHSE, but not DSE or HCSE, inhibited spawning (P less than 0.01) in 36% of the exposed fish and hepatic AHH activity in the non-spawning fish was significantly (P less than 0.05) higher than in the fish that did spawn. These results suggest a potential for reproductive toxicity in benthic fish after exposure to sediment-associated contaminants.     

Southern flounder hepatic and intestinal metabolism and DNA binding of benzo[a]pyrene (BaP) metabolites following dietary administration of low doses of BaP, BaP-7,8-dihydrodiol or a BaP metabolite mixture

Certain finfish species living in chemically polluted environments exhibit a high incidence of gastrointestinal tract tumors. Carnivorous fish in such environments are likely to consume invertebrates which contain chemical procarcinogens and the invertebrate biotransformation products of these compounds. The retention in tissues, extent of DNA adduct formation in liver and intestine, and metabolite composition of bile was investigated in southern flounder following gavage with pure [3H]- or [14C]benzo[a]pyrene (BaP), pure [14C]benzo[a]pyrene-7,8-dihydrodiol (BaP-7,8D), or hepatopancreas from spiny lobsters previously dosed with [3H]- or [14C]BaP (Metab.HP). Metab.HP contained mainly polar conjugates of BaP diols, triols and tetraols. BaP-7,8D was retained in fish tissues and bile at 24 h to a greater extent (33.6% of the dose), than either BaP (19.00%) or Metab.HP (6.6%). Hepatic and intestinal DNA isolated from all dosed fish contained covalently bound radioactivity, but exposure to BaP-7,8D or BaP resulted in significantly higher binding in both tissues than exposure to Metab.HP. Hepatic DNA from BaP and BaP-7,8D-dosed flounder contained 0.24 +/- 0.07 and 0.33 +/- 0.06 pmol BaP equivalents/mg DNA respectively (mean +/- S.E.), while hepatic DNA isolated from Metab.HP-dosed flounder contained 0.006 +/- 0.002 pmol BaP equivalents/mg DNA. Binding of radioactivity to intestinal DNA was significantly higher than to hepatic DNA for flounder dosed with Metab.HP (0.026 +/- 0.003) or with BaP (0.76 +/- 0.27) but not for flounder dosed with BaP-7,8D (0.44 +/- 0.09). These studies show that dietary BaP, and metabolites likely to be present in invertebrates, can be absorbed by the southern flounder and form DNA adducts in target organs.     

DNA adduct formation and persistence in liver and extrahepatic tissues of northern pike (Esox lucius) following oral exposure to benzo[a]pyrene, benzo[k]fluoranthene and 7H-dibenzo[c,g]carbazole.

The formation and persistence of DNA adducts in liver, intestinal mucosa, gills and brain of juvenile northern pike (Esox lucius) following oral exposure to benzo[a]pyrene (BaP), benzo[k]fluoranthene (BkF) and 7H-dibenzo[c,g]carbazol (DBC) were analysed by 32P-postlabelling. The dosage was 25 micromol/kg body weight of each substance, administered on 5 occasions with an interval of 12-14 days. Sampling was carried out 9 days after the second treatment, and 9, 16, 33 and 78 days after the fifth treatment. Pikes were also fed with the substances singly for comparison of adduct patterns. A complex pattern of adducts was detected in all examined tissues from fish treated with the mixture. Total adduct levels were highest in intestine (347+/-17.4 nmol adducts/mol nucleotides, mean+/-SE), followed by liver (110+/-9.3), gills (69+/-6) and brain (14+/-4.2). In pike treated with BaP alone, one major adduct was detected in all examined tissues. This BaP-adduct made up approximately 50% of the total amount of adducts in the brain. Corresponding values in liver, intestine and gills were 23, 31 and 34%, respectively. One relatively weak BkF-adduct and at least 10 different DBC-adducts were detected in all analysed tissues. Total adduct level in the intestine declined to 29.4% of the maximum value 78 days after the last exposure, while there was no significant decline in adduct levels in liver, gills or brain. The results suggest that intestine is more susceptible to adduct formation than liver after oral exposure, and that adduct levels in the intestine represent ongoing or relatively recent exposure. DNA adducts in the other investigated tissues were much more persistent and may therefore accumulate during long-term exposure.     

Establishing the Causal Relationship between Polycyclic Aromatic Hydrocarbon (PAH) Exposure and Hepatic Neoplasms and Neoplasia-Related Liver Lesions in English Sole (Pleuronectes vetulus)

For almost 25 years our laboratory has studied the impact of PAHs and related industrial contaminants on benthic fish, following an interdisciplinary approach involving chemical exposure assessment linked to synoptic detection of various effects at several levels of biological organization. These data demonstrate a cause-and-effect relationship between neoplastic and neoplasia-related liver lesions in English sole, and exposure to PAHs, and to a lesser degree, chlorinated hydrocarbons such as PCBs. In statistical analyses of data from multiple field studies conducted since 1978, exposure to PAHs measured in various compartments has consistently been identified as a highly significant, major risk factor for neoplasms and related lesions in this species, with PCB exposure shown to be a significant, but less consistent and less strong risk factor for these lesions. A cause-and-effect relationship between PAHs and toxicopathic liver lesions in this species is further supported by the experimental induction of toxicopathic lesions identical to those observed in field-collected fish, in sole exposed in the laboratory to model carcinogenic PAHs such as BaP or to PAH-rich extracts of sediments from Eagle Harbor, a severely PAH-contaminated site in Puget Sound. More recent field studies have identified significant associations between hepatic cytochrome P4501A (CYP1A) induction and xenobiotic-DNA adduct formation, and hepatic lesion prevalences in wild subadult English sole. Field studies in Eagle Harbor subsequent to capping of the most PAH-contaminated region of this harbor with clean dredge spoils have shown a decline in exposure to PAHs as assessed by biliary fluorescent aromatic compounds (FACs) and hepatic xenobiotic-DNA adducts. This decline in PAH exposure has been accompanied by a dramatic decline in risk of occurrence of toxicopathic hepatic lesions in English sole from Eagle Harbor. Further, laboratory studies have induced lesions in English sole by injections of extracts from PAHcontaminated sediments. Overall, these findings relating to exposure to PAHs and chlorinated hydrocarbons and the occurrence of hepatic neoplasms and neoplasiarelated lesions in English sole fulfill the classic criteria for causality in epizootiological or ecological risk assessment studies, including: (1) strength of association, (2) consistency of association, (3) specificity of association, (4) toxicological and biological plausibility, (5) temporal sequence/timing (i.e., exposure precedes disease, effect decreases when the cause is decreased or removed), (6) dose-response or biological gradient, and (7) supportive experimental evidence.     

Monitoring the exposure of rats to 2-acetylaminofluorene by the estimation of mutagenic activity in excreta, sister-chromatid exchanges in peripheral blood cells and DNA adducts in peripheral blood, liver and spleen

The sensitivity of various methods suitable for biomonitoring the exposure to genotoxicants was compared in an animal model. The results were related to the presence of genotoxic effects in the target organ. Groups of male Wistar rats were given one oral dose of 0, 0.1, 10 or 200 mg 2-acetylaminofluorene (2-AAF)/5 ml dimethyl sulphoxide/kg body weight. Peripheral blood cells, excreta, liver and spleen were collected at different time intervals after dosing. Mutagenicity in urine and extracts of faeces was determined using the Ames test with Salmonella typhimurium TA98 with and without S9 and with and without beta-glucuronidase. Genotoxic effects were studied by measuring DNA-adduct formation in lymphocytes, liver and spleen, and sister-chromatid exchanges (SCEs) in lymphocytes. DNA adducts were measured with immunochemical techniques and postlabelling methods. Mutagenicity in urine and faeces, collected during the first 24 h after treatment, was detected at 2-AAF doses of 1 mg/kg b.w. and higher. At these doses DNA adducts also became apparent in the liver, the main target organ for tumour induction by 2-AAF. The adduct detected appeared to be the N-(deoxyguanosin-8-yl)-2-AAF adduct. There was no evidence of the presence of any other types of DNA adducts. At doses of 1 and 10 mg/kg b.w. no mutagenicity was detected in excreta collected during the second and third day after dosing. The DNA-adduct level in liver cells of the 1 mg/kg b.w. group was maximal 24 h after dosing. At 200 mg/kg b.w. a delay in excretion of mutagenicity with urine and faeces was seen and at 10 and 200 mg/kg b.w. the amount of DNA adducts continued to increase with time after dosing. At 24 and 48 h after treatment with 10 mg, the adduct levels were of the same order of magnitude as those found after the 20-fold higher dose. This points to overloading of the metabolizing system which in combination with the enterohepatic circulation, may lead to an increased retention of 2-AAF in the body. A slightly increased incidence of SCEs of doubtful significance was seen in lymphocytes, but only at the very high dose of 200 mg/kg b.w. No DNA adducts could be detected in blood lymphocytes or spleen cells at any of the dose levels applied, either with the immunochemical or with the postlabelling method.(ABSTRACT TRUNCATED AT 400 WORDS)     

Relative sensitivity of 32P-postlabelling of DNA and the autoradiographic UDS assay in the liver of rats exposed to 2-acetylaminofluorene (2AAF)

Groups of male Alderley Park rats were dosed concomitantly with 2-acetylaminofluorene (2AAF) by gavage at doses between 0.01 mg/kg and 40 mg/kg, and livers sampled 2-72 h later. The liver of one group of animals was perfused to yield hepatocytes which were assayed in vitro for unscheduled DNA synthesis (UDS) via incorporation of tritiated thymidine and autoradiography. DNA was extracted from the livers of the other group and DNA adduct levels determined using the 32P-postlabelling technique. The major C-8 2-aminofluorene/guanosine adduct and 3 minor adducts were quantitated, enabling the relative sensitivity of the 2 techniques to be compared. A dose- and time-related UDS response was observed, which, at the most sensitive time-point (12 h) enabled DNA repair to be discerned at a dose level of 0.1-1 mg/kg of 2AAF, a response classified as formally positive at 5 mg/kg 2AAF. Only the C-8 adduct, as determined by 32P-postlabelling, was discernible at 0.01 mg/kg of 2AAF, although other adducts were visible on autoradiograms at higher dose levels. It is concluded that as part of a well-defined dose response, UDS can be discerned with confidence for doses of 2AAF between approximately 0.1 and 5 mg/kg, and DNA adducts for doses of 2AAF between approximately 0.01 and 1 mg/kg. Discernible UDS for 2AAF in the rat liver is apparent at approximately 13 DNA (total) adducts/10(8) nucleotides, or approximately 8 DNA (C-8) adducts/10(8) nucleotides. The presumed C-8 2-acetylaminofluorene/guanosine adduct, prepared by reaction of 2-acetoxy-2-acetylaminofluorene (2AAAF) with DNA, was a significant but unreliable marker of 2AAF/DNA adducts in the rat liver in vivo. DNA repair did not appear to remove DNA adducts selectively, and adducts remained in DNA when discernible DNA repair had ceased.     

Quantification of aristolochic acid-derived DNA adducts in rat kidney and liver by using liquid chromatography-electrospray ionization mass spectrometry

Aristolochic acid (AA), derived from the herbal genus Aristolochia and Asarum, has recently been shown to be associated with the development of nephropathy. Upon enzyme activation, AA is metabolized to the aristolactam-nitrenium ion intermediate, which reacts with the exocyclic amino group of the DNA bases via an electrophilic attack at its C7 position, leading to the formation of the corresponding DNA adducts. The AA-DNA adducts are believed to be associated with the nephrotoxic and carcinogenic effects of AA. In this study, liquid chromatography coupled with electrospray ionization mass spectrometry (LC-MS) was used to identify and quantify the AA-DNA adducts isolated from the kidney and liver tissues of the AA-dosed rats. The deoxycytidine adduct of AA (dC-AA) and the deoxyadenosine-AA adduct (dA-AA) were detected and quantified in the tissues of rats with one single oral dose (5mg or 30mg AA/kg body weight). The deoxyguanosine adduct (dG-AA), however, was detected only in the kidney of rats that were dosed at 30mg AA/kg body weight for three consecutive days. The amount of AA-DNA adducts found in the rats correlated well with the dosage.     

苯并（a）芘对大弹涂鱼肝细胞超微结构的影响

在实验生态条件下,研究不同浓度苯并(a)芘(BaP)暴露下大弹涂鱼肝脏细胞超微结构的变化。结果表明,暴露于低浓度(0．5mg·L^-1)BaP 7d,大弹涂鱼肝脏细胞内的细胞器受到不同程度的损伤,其中线粒体和内质网是受BaP暴露影响最明显的细胞器,细胞核也受到不同程度的影响,细胞质中脂滴也增加;而暴露于高浓度(5mg·L^-1)BaP 2h,不仅是线粒体和内质网,几乎所有细胞器都受到严重影响,细胞器严重退化,细胞结构遭到严重破坏。研究结果证实,BaP可对大弹涂鱼肝细胞内多种细胞器造成损伤,并且BaP浓度越高,损伤程度越严重。     

Oxidative DNA damage in tissues of English sole (Parophrys vetulus) exposed to nitrofurantoin

Juvenile English sole were exposed intramuscularly to nitrofurantoin (NF) and the levels of 8-hydroxy-2′deoxyguanosine (8-OH-dG) in liver, kidney and blood were determined using reversed-phase HPLC with electrochemical detection. Identification and quantitation of the 8-OH-dG in the samples was accomplished by comparison with standard 8-OH-dG, which was characterized by UV spectroscopy and fast-atom bombardment mass spectrometry. The levels of hepatic 8-OH-dG increased (r² = 0.59, P = 0.015) with the dose of NF (0.10 – 10 mg NF/kg fish). In kidney and blood, however, the levels of 8-OH-dG were significantly higher than controls only at the highest dose tested. The level of binding in liver ranged from 0.37 to 0.76 fmol 8-OH-dG/μg DNA. The levels of hepatic 8-OH-dG reached a maximum (approx. 1 fmol 8-OH-dG/μg DNA) between 1 and 3 days after exposure, followed by a decrease to control levels (approx. 0.25 fmol 8-OH-dG/μg DNA) at 5 days post-exposure. These data demonstrate the first direct evidence for the formation of oxidized DNA bases resulting from the metabolism of a nitroaromatic compound by fish.     

Relative sensitivity of 32P-postlabelling of DNA and the autoradiographic UDS assay in the liver of mice exposed to 2-acetylaminofluorene (2AAF)

In contrast to earlier studies conducted at lower dose levels, 2AAF is shown to induce a positive UDS response in the liver of mice dosed orally at dose levels between 500 and 1000 mg/kg. Similarly exposed mice had low levels of 2AAF-related hepatic DNA adducts at dose levels in the range 10-1000 mg/kg 2AAF, as determined by 32P-postlabelling analysis. It is concluded that the attenuated UDS response observed in the mouse liver, as compared to the rat liver, is due primarily to metabolic differences between these two species, coupled to a reduced capacity for UDS in the mouse liver for a given level of total 2AAF-related adducts per unit of DNA. These observations are compared and contrasted with identical studies conducted in the rat and reported in the preceding paper (Gallagher et al., 1991).     

Benzo[a]pyrene-induced toxicity: paradoxical protection in Cyp1a1(-/-) knockout mice having increased hepatic BaP-DNA adduct levels.

Previous studies have shown that cytochrome P450 1A1 (CYP1A1), CYP1B1, and prostaglandin-endoperoxide synthase (PTGS2) are inducible by benzo[a]pyrene (BaP) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin), and all three metabolize BaP to reactive DNA-binding intermediates and excreted products. Because these three enzymes show differing patterns of basal levels, inducibility, and tissue-specific expression, animal studies are necessary to delineate the role of CYP1A1 in BaP-mediated toxicity. In mice receiving large daily doses of BaP (500 mg/kg i.p.), Cyp1a1(-/-) knockout mice are protected by surviving longer than Cyp1a1(+/-) heterozygotes. We found that a single 500 mg/kg dose of BaP induces hepatic CYP1A1 mRNA, protein, and enzyme activity in Cyp1a1(+/-) but not in Cyp1a1(-/-) mice; TCDD pretreatment increases further the CYP1A1 in Cyp1a1(+/-) but not Cyp1a1(-/-) mice. Although a single 500 mg/kg dose of BaP was toxic to Cyp1a1(+/-) mice (serum liver enzyme elevated about 2-fold above control levels at 48 h), Cyp1a1(-/-) mice displayed no hepatotoxicity. Unexpectedly, we found 4-fold higher BaP-DNA adduct levels in Cyp1a1(-/-) than in Cyp1a1(+/-) mice; TCDD pretreatment lowered the levels of BaP-DNA adducts in both genotypes, suggesting the involvement of other TCDD-inducible detoxification enzymes. BaP was cleared from the blood much faster in Cyp1a1(+/-) than Cyp1a1(-/-) mice. Our results suggest that absence of the CYP1A1 enzyme protects the intact animal from BaP-mediated liver toxicity and death, by decreasing the formation of large amounts of toxic metabolites, whereas much slower metabolic clearance of BaP in Cyp1a1(-/-) mice leads to greater formation of BaP-DNA adducts.     

Effect of bromobenzene and O-bromophenol on kidney and liver of English sole (Parophrys vetulus)

1. English sole (Parophrys vetulus) were injected intraperitoneally with a single dose of 9.8 mmol bromobenzene/kg of fish or 1.9 mmol O-bromophenol/kg of fish, both known renal toxicants in mammals. 2. Kidney, liver, gill spleen, intestines, heart and blood samples were subsequently obtained up to 48 hr post-injection for determination of microscopic lesions, concentrations of selected tissue antioxidants (glutathione and ascorbic acid), and selected serum parameters. 3. Bromobenzene and O-bromophenol were both found to be hepatotoxic in English sole, as indicated by the presence of hepatocellular coagulation necrosis and fatty change in the liver, altered glutathione and ascorbic acid levels in liver tissue, elevated serum aspartate aminotransferase and alkaline phosphatase activity and increased serum glucose and triglyceride levels. 4. No evidence of nephrotoxicity was found in English sole exposed to either toxicant. 5. It is concluded that bromobenzene and O-bromophenol cannot be used as model nephrotoxicants but can be used as hepatotoxicants in English sole.     

2-(Allylthio)pyrazine inhibition of aflatoxin B1-induced hepatocarcinogenesis in rats.

2-(Allylthio)pyrazine (2-AP), a synthetic pyrazine derivative with an allylsulfur moiety, has hepatoprotective effects against toxicants. Effect of 2-AP on hepatic tumorigenesis in association with glutathione S-transferase (GST) induction was examined in rats exposed to aflatoxin B1 (AFB1). Both AFB1-DNA adduct formation in the liver and urinary elimination of 8,9-dihydro-8-(N7-guanyl)-9-hydroxy-aflatoxin B1 (AFB1-N7-guanine) adduct were also determined. Male Sprague Dawley rats were treated with 2-AP at the daily oral doses of 10, 25 and 50 mg/kg for 16 consecutive days, during which four repeated doses of AFB1 (1.0 mg/kg) were given to the animals. Rats were then subjected to two-thirds of hepatectomy, followed by administration of phenobarbital (PB). Focal areas of hepatocellular alteration were identified after 44 days and preneoplastic foci expressing the placental form of glutathione S-transferase P (GST-P) were quantified by immunostaining of liver sections. 2-AP reduced the volume of liver occupied by GST-P foci by 65-96%. Under these experimental conditions, 2-AP treatment resulted in significant elevations in GST activity in the liver. Levels of radiolabeled AFB1 covalently bound to hepatic DNA, RNA and proteins were significantly reduced in rats treated with 2-AP for 5 days. 2-AP pretreatment also caused a 45% reduction in the urinary elimination of AFB1-N7-guanine adduct over the 24-h postdosing period. The present findings demonstrated that 2-AP exhibited protective effects against AFB1-induced hepatocarcinogenesis in rats with a marked decrease in the level of AFB1-DNA adduct. Reduction of hepatic DNA adducts might result from elevations of activity of GST, which catalyzes detoxification of the carcinogen.     

Analysis of DNA and protein adducts of benzo[a]pyrene in human tissues using structure-specific methods

We review studies which investigate the presence, using structure-specific analytical methods, of DNA or protein adducts of the carcinogen benzo[a]pyrene (BaP) in human tissues. The analytical methods include high performance liquid chromatography with fluorescence detection and gas chromatography-mass spectrometry. Although, for DNA detection these methods are somewhat less sensitive than non-specific techniques such as 32P-postlabeling and immunoassay, they have the distinct advantage of providing reliable structural information. In order to achieve adequate sensitivity, these methods often require the use of fairly large amounts of DNA (>100 microg) or protein (50-100mg). Most studies reviewed here measured tetraols released from DNA or protein by hydrolysis of adducts derived from (7R,8S)-dihydroxy-(9S,10R)-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE), a major ultimate carcinogen of BaP. BPDE-DNA adducts were detected in 39% of 705 samples analyzed. BPDE-protein adducts were found in 59% of 772 samples. There was no single exposure situation that led to an overwhelming presence of detectable adducts. For example, BPDE-DNA adducts were detected in 45% of smokers, 33% of former smokers, 52% of non-smokers, 39% of occupationally exposed individuals, and 34% of environmentally exposed people. Adduct levels were influenced by polymorphisms in carcinogen metabolizing genes such as GSTM1, the presence of which was frequently protective. The relatively high occurrence of non-detectable adducts may result from low levels of BaP exposure and host factors such as genetic polymorphisms. Our analysis demonstrates that the presence of BaP adducts in human tissues cannot be assumed, even in situations where exposure to BaP is relatively high.     

In vivo covalent binding of [14C]trinitrotoluene to proteins in the rat.

When a single dose of [14C]trinitrotoluene was administered intraperitoneally (i.p.) to rats at 1, 10 or 50 mg/kg of body weight, covalently bound radioactivity was detected in globin, plasma proteins and proteins in the liver and kidney. The extent of covalent binding was dose dependent and was highest in plasma and renal proteins at all times up to 4 h after dosing. Covalent adduct levels in globin, however, decline slower than others. At a dose of 50 mg/kg of body weight, globin covalent adduct levels peaked at 1 h after dosing at 182 pmol/mg protein and subsequently decreased to approximately 50 pmol/mg protein between days 1 and 8. Of the covalent adduct levels in liver and kidney, those in the 10,000 x g and microsomal fractions were found to be higher than that in the cytosolic fraction. Radioactivity covalently bound to globin and the hepatic proteins was susceptible to dilute acid hydrolysis from which 2-amino-4,6-dinitrotoluene (2A) and 4-amino 2,6-dinitrotoluene (4A) were the major products recovered by solvent extraction. Upon acetylation, the hydrolysate gave rise to derivatives identified as the acetates of 2A and 4A on the basis of mass spectrometry and HPLC cochromatography with authentic samples. Four hours after an i.p. dose of [14C]TNT at 50 mg/kg of body weight about 0.4% of the dose was found as bound adducts to hemoglobin, of which approximately 48% was recovered as solvent extractable radioactivity after acid hydrolysis. About 2% of the radioactive dose was in the liver, of which approximately 30% was covalently bound to hepatic proteins, and approximately 49% of that was convertible to solvent extractable radioactivity upon acid hydrolysis. In vitro incubation of [14C]TNT with blood showed that there was a linear increase of covalent adducts in globin during the first 2 h of incubation; the concentration of covalent adducts was slightly higher than that with plasma proteins. The major compounds recovered from the hydrolysate of the globin adducts were also 2A and 4A as obtained from globin in the in vivo studies. On the basis of the in vitro and in vivo study results, we have confirmed the formation of protein adducts following a single i.p. administration of [14C]TNT at 1, 10 or 50 mg/kg of body weight to the rat or by in vitro incubation with blood.(ABSTRACT TRUNCATED AT 400 WORDS)     

Oxidative DNA damage in tissues of English sole (parophrys vetulus) exposed to nitrofurantoin

Juvenile English sole were exposed intramuscularly to nitrofurantoin (NF) and the levels of 8-hydroxy-2′deoxyguanosine (8-OH-dG) in liver, kidney and blood were determined using reversed-phase HPLC with electrochemical detection. Identification and quantitation of the 8-OH-dG in the samples was accomplished by comparison with standard 8-OH-dG, which was characterized by UV spectroscopy and fast-atom bombardment mass spectrometry. The levels of hepatic 8-OH-dG increased (r² = 0.59, P = 0.015) with the dose of NF (0.10 – 10 mg NF/kg fish). In kidney and blood, however, the levels of 8-OH-dG were significantly higher than controls only at the highest dose tested. The level of binding in liver ranged from 0.37 to 0.76 fmol 8-OH-dG/μg DNA. The levels of hepatic 8-OH-dG reached a maximum (approx. 1 fmol 8-OH-dG/μg DNA) between 1 and 3 days after exposure, followed by a decrease to control levels (approx. 0.25 fmol 8-OH-dG/μg DNA) at 5 days post-exposure. These data demonstrate the first direct evidence for the formation of oxidized DNA bases resulting from the metabolism of a nitroaromatic compound by fish.     

DNA adducts as a biomarker of polycyclic aromatic hydrocarbon exposure in aquatic organisms: relationship to carcinogenicity

The use of DNA adduct measurement as a biomarker of exposure to polycyclic aromatic hydrocarbons (PAHs) is now well established in ecotoxicology. In particular, DNA adduct levels in aquatic organisms has been found to produce a better correlation with PAH exposure than PAH concentrations in organisms. DNA adducts levels are most commonly determined using the ³²P-postlabelling assay which measures total aromatic adducts. The relationship between relative DNA adduct formation and carcinogenicity has been investigated for a number of carcinogenic and non-carcinogenic PAHs using an in vitro system. Our results demonstrate that relatively high levels of DNA adducts can be produced by some non-carcinogenic PAHs, while other non-carcinogenic compounds do not produce detectable adducts. In addition, it has been shown that all carcinogenic PAHs investigated produce DNAadducts and that a relationship exists between relative adduct formation and carcinogenic potency. An investigation of adduct levels in fish liver and crustacean hepatopancreas in Oxley Ck, Brisbane has shown that higher than expected DNA adduct levels were correlated with the presence of carcinogenic and non-carcinogenic PAHs with high relative adduct forming potential.     

Benzo(a)pyrene diolepoxide-haemoglobin and albumin adducts at low levels of benzo(a)pyrene exposure

A biomonitoring study was conducted to simultaneously measure individual benzo(a)pyrene (BaP) exposure in 50 office employees, not occupationally exposed to polycyclic aromatic hydrocarbons (PAH), using personal samplers and the formation of (+) r-7, t-8-dihyroxy-t-9,t-10-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (BPDE) adducts to haemoglobin (BPDE-Hb) and serum albumin (BPDE-SA). The population enrolled was exposed to an average of 0.58 ± 0.46 ng BaP m^-3 (mean ± SD). The concentration of BaP collected from smokers' samples was double that from non-smokers (P = 0.007). BPDE adducts to Hb and SA were quantified as BaP tetrols released from hydrolysis of macromolecules and measured by high-resolution gas chromatography-negative ion chemical ionization-mass spectrometry. BPDE-Hb adducts were detected in 16% of the population and BPDE-SA adducts in 28%. Smoking did not affect adduct formation. When BaP personal monitoring data were used as the criterion of exposure, no correlation was found with the presence and the levels of BPDE-Hb and BPDE-SA adducts. Undetected sources of PAH, such as the diet, might markedly alter the exposure profile depicted by individual air sampling and affect the frequency and levels of protein biomarkers. This is the first comparative analysis of BPDE-Hb and BPDE-SA adducts, providing reference values for these biomarkers in a general urban population. However it is difficult to establish which biomarkers would be the more relevant in assessing low BaP exposure, due to undetectable factors such as dietary PAHs, that might have influenced the results to some degree.