Levels Of Hepatic DNA Adducts Research Articles

Hepatic DNA damage in harbour porpoises (Phocoena phocoena) stranded along the English and Welsh coastlines.

One level at which persistent organic pollutants (POPs) and polycyclic aromatic hydrocarbons PAHs) can exert damage is by causing DNA strand‐breaks or nucleotide base modifications, which, if unrepaired, can lead to embryonic mutations, abnormal development and cancer. In marine ecosystems, genotoxicity is expected to be particularly strong in long‐lived apex predators due to pollutant bioaccumulation. We conducted 32P‐postlabeling analyses optimized for the detection and quantification of aromatic/hydrophobic DNA adducts in the livers of 40 sexually‐mature North Atlantic harbour porpoises (Phocoena phocoena) stranded along the English and Welsh coastlines. We examined hepatic tissue to search for inflammatory and preneoplastic lesions and examine their association with adduct levels. Adducts were found in all porpoises (mean: 17.56 ± 11.95 per 108 nucleotides), and were higher than levels reported for marine vertebrates from polluted sites. The pollutants causing the induced DNA adducts could not be further characterized. Hepatic DNA damage did not correlate with levels of blubber POP concentrations (including total polychlorinated biphenyl [PCBs], dichlorodiphenyltrichloroethane [DDT] and dieldrin); PAH concentrations were not available for the present study. However, DNA damage predicted occurrence of inflammatory and preneoplastic lesions. Further, our data showed a reduction in hepatic DNA adduct levels with age in the 40 animals examined while POP concentrations, particularly PCBs, increased with age. Using a different dataset of 145 mature male harbour porpoises confirmed that higher contaminant levels (total PCBs, DDT and dieldrin) are found in older animals. The reduction in hepatic DNA adduct levels in older animals was in accordance with other studies which show that suppression of hepatic CYP1A enzyme activity at high PCB concentrations might impact on CYP1A‐mediated DNA adduct formation of PAHs which are ubiquitous environmental pollutants and readily metabolized by CYP1A to species binding to DNA. In summary, our study shows that pollutant‐induced DNA damage is prevalent in harbour porpoises from UK waters and may lead to detectable sub‐lethal hepatic damage. Environ. Mol. Mutagen. 59:613–624, 2018. © 2018 The Authors Environmental and Molecular Mutagenesis published by Wiley Periodicals, Inc. on behalf of Environmental Mutagen Society

Abundance of DNA adducts of methyleugenol, a rodent hepatocarcinogen, in human liver samples

Methyleugenol is a genotoxic carcinogen in mice and rats, the liver being the primary target tissue. Methyleugenol occurs in fennel and many herbs and spices. Furthermore, methyleugenol-containing plant extracts and chemically prepared methyleugenol are used as flavoring agents. We analyzed surgical human liver samples from 30 subjects for the presence of DNA adducts originating from methyleugenol using isotope-dilution ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Twenty-nine samples unambiguously contained the N (2)-(trans-methylisoeugenol-3'-yl)-2'-deoxyguanosine adduct. A second adduct, N (6)-(trans-methylisoeugenol-3'-yl)-2'-deoxyadenosine, was also found in most samples, but at much lower levels, in agreement with the results from experimental models. The maximal and median levels of both adducts combined were 37 and 13 per 10(8) nucleosides (corresponding to 4700 and 1700, respectively, adducts per diploid genome). This is the first demonstration of DNA adducts formed by a xenobiotic in human liver using UPLC-MS/MS, the most reliable method available. It has been estimated for diverse rat and mouse hepatocarcinogens that 50-5500 adducts per 10(8) nucleosides are present after repeated treatment at the TD50 (daily dose that halves the probability to stay tumor-free in long-term studies). We conclude that the exposure to methyleugenol leads to substantial levels of hepatic DNA adducts and, therefore, may pose a significant carcinogenic risk.

Screening of hydrophobic DNA adducts in flounder ( Platichthys flesus) from the Baltic Sea

Neoplasia and other histopathological lesions in flounder ( Platichthys flesus) liver have been investigated in several European sea areas, including the Baltic Sea. Several studies have been able to link neoplasm epizootics in fish with the exposure to genotoxins such as polycyclic aromatic hydrocarbons (PAHs). The level of hydrophobic DNA adducts in tissue DNA reflects the exposure of the organism to PAHs. Using hydrophobic DNA adduct levels as biomarkers, possible PAH exposure was assessed in flounder from 10 different sites in the Baltic Sea, collected during the years 1995–1997. The results show that the overall levels of hepatic DNA adducts were low and, in general, the chromatograms appeared clean. The highest levels of DNA adducts were found at two sites in the southern Baltic Sea. There were no statistically significant differences in adduct levels between the sites. Our results indicate that flounder from studied off shore sites of the Baltic Sea had not been exposed to a greater extent to large polycyclic hydrophobic hydrocarbons in their environment.

DNA adduct analysis and histopathological biomarkers in European flounder ( Platichthys flesus) sampled from UK estuaries

The presence of genotoxic and potentially carcinogenic chemical contaminants in the estuarine and coastal marine environment is well documented. In this study, European flounder ( Platichthys flesus) sampled from eight UK estuaries were analysed for hepatic DNA adducts, using the 32 P -postlabelling assay and liver histopathology as part of an on going survey to establish the health status of UK estuaries. Fish were collected from the estuaries Tyne, Mersey, Thames, Alde (reference site), Belfast, Forth, Clyde and Southampton. At the majority of contaminated sites (Southampton, Thames, Clyde, Tyne and Mersey) the predominant DNA adduct profile consisted of diagonal radioactive zones (DRZs). In contrast, flounder collected from the Forth, Alde and Belfast lacked DRZs with only background levels of DNA damage being observed. Statistically significant differences were observed between several of the sites sampled with the hepatic DNA adduct levels detected in flounder from Southampton, Thames and Clyde statistically elevated ( P < 0.05) over those levels detected at the Tyne (Southampton and Thames only), Forth, Alde and Belfast. Histological analysis of these samples revealed a range of lesions including foci of cellular alteration, hepatocellular fibrillar inclusions, nuclear pleomorphisms along with non-toxicopathic changes/alterations, such as those associated with cell turnover (apoptosis, necrosis, regeneration) and immune-related functions (melanomacrophage aggregates, inflammation). Although it is difficult to associate higher prevalence of these lesion types with specific sites, generally, the lowest prevalence was seen in flounder captured from the Alde estuary, with higher prevalence (particularly of melanomacrophage aggregates, inflammation and necrotic foci) seen in fish from the contaminated sites.

Ethanol Enhances the Activation of 1-Hydroxymethylpyrene to DNA Adduct-Forming Species in the Rat

1-Methylpyrene, a hepatocarcinogen, is metabolized via 1-hydroxymethylpyrene (HMP) to a reactive sulfuric acid ester (SMP). SMP forms DNA adducts and, alternatively, is conjugated with glutathione, leading to the excretion of methylpyrenyl mercapturic acid (MPMA). However, 1-pyrenylcarboxylic acid and its derivatives constitute nearly 80% of the excreted metabolites after administration of HMP to rats. Thus, side-chain oxidation, probably catalyzed by alcohol and aldehyde dehydrogenases, competes with the activation to SMP. We therefore studied the bioactivation of HMP in rats treated with 4-methylpyrazole (an inhibitor of alcohol dehydrogenases), disulfiram (an inhibitor of aldehyde dehydrogenases), or ethanol (a substrate of alcohol dehydrogenases). These metabolic modulators enhanced the levels of hepatic DNA adducts 28-, 3.8-, and 15-fold, respectively. Exceptionally high levels of adducts, up to ∼1 per 10 4 nucleotides, were observed. The metabolic modulators also raised the excretion of MPMA, a possible biomarker for the formation of ultimate carcinogen. Thus, interaction with other chemicals may dramatically affect the toxicological effects of some polycyclic aromatic carcinogens. In particular, the effect of ethanol may be relevant for some human exposures to polycyclic aromatic compounds.

DNA adducts in liver and leukocytes of flounder ( Platichthys flesus) experimentally exposed to benzo[ a]pyrene

In the present study the levels of hydrophobic DNA adducts detected by 32P-postlabelling were followed in liver and leukocytes of flounder ( Platichthys flesus) over 10 days following single i.p. injections of two doses of BaP (10 and 50 mg kg −1 fish weight, respectively). DNA adducts were detected in both tissues of exposed fish 2 days post injection and continued to rise on day 5 and day 10. In flounder exposed to the lower dose of BaP, the levels of hepatic DNA adducts reached higher values on the fifth day compared with flounder exposed to the higher dose. However, at the end of the experiment, the DNA adduct level was again higher in fish from the high dose group compared with the low dose group. There was no substantial increase of DNA adducts in liver of flounder from the low dose group after day 5, while the adduct levels in flounder liver from the high dose group increased throughout the experiment. Earlier studies detecting DNA adducts in BaP-exposed flatfish with the 32P-postlabelling technique have reported declining adduct levels from about 2 days after the exposure, regardless of exposure route. In contrast, the results from our study did not confirm a rapid increase and successive decline of hydrophobic adducts in liver of BaP-exposed flounder.

DNA adducts in perch ( Perca fluviatilis) from a creosote contaminated site in the River Ångermanälven, Sweden

Levels of hepatic DNA adducts, analysed by 32P-postlabelling, histopathological lesions and organosomatic indices, were measured in perch ( Perca fluviatilis). The fish were caught at five sites in the river Ångermanälven, Sweden: one site known to be contaminated with creosote, sites downstream from the contaminated site and a reference site upstream. Perch were also caught at a long-distance reference site. The level of DNA adducts in fish from the creosote-contaminated site was 6.8±4.1 nmol mol −1 nucleotides compared to 0.21±0.21 nmol mol −1 nucleotides in fish from the long-distance reference site. The adduct level was also significantly increased compared to adduct levels in fish from the local reference site. Hepatocellular degeneration and macrophage aggregates were observed but did not correlate with a specific site in the river. No effects on organosomatic indices that correlated with distance from the contaminated site were observed. In the laboratory, perch were exposed to an organic solvent extract prepared from sediment collected at the creosote-contaminated site or to benzo(a)pyrene (BaP) by oral administration. Perch treated with the extract had adduct patterns very similar to those observed in perch from the contaminated field site. Two diagonal zones including 10–12 adduct spots were observed on the autoradiograms. One adduct was tentatively identified by co-chromatography with a 32P-labelled standard of 7 R,8 S,9 S-trihydroxy, 10 R-( N 2-deoxyguanosyl 3′-phosphate)-7,8,9,10-tetrahydrobenzo(a)pyrene (BaPDE-dG-3′p) in two different solvent systems. Autoradiograms derived from the BaP-exposed perch showed one major adduct and several less intense spots. The major BaP adduct had the same chromatographic properties as the standard BaPDE-dG-3′p adduct. This study shows that 32P-postlabelling analysis of DNA adducts in perch can be used as a sensitive indicator of exposure to genotoxic polycyclic aromatic hydrocarbons, and that comparison of DNA adduct patterns of fish exposed in the field and laboratory can be used to determine the responsible source of pollutants.

Dose-response relationships for hepatic aflatoxin B1-DNA adduct formation in the rat in vivo and in vitro: the use of immunoslot blotting for adduct quantitation.

An immunoslot blotting (ISB) method for quantitating aflatoxin B1-DNA adduct levels has been developed and used to examine the relationship between dose and hepatic aflatoxin B1-DNA adduct levels in rats fed aflatoxin B1 (AFB1) in the diet at dose levels of between 0.5 and 10 micrograms/kg/day. The method has also been used to examine the dose-response relationship for adduct formation in precision-cut rat liver slices incubated with AFB1 at concentrations between 0.01 and 2 microM. For the feeding studies, groups of male Fisher F344 rats were given AFB1 in the diet for periods of 1 to 10 weeks and hepatic DNA adduct levels determined using ISB. The time for adduct levels to reach steady-state conditions was determined in animals given approximately 10 micrograms of AFB1/kg/day and steady-state levels at lower concentrations measured. The time course for the accumulation of AFB1-DNA adducts in rat liver slices incubated with AFB1 at 0.5 microM has been investigated and the relationship between adduct formation and AFB1 concentration over a wide concentration range in liver slices has been determined.

Formation of DNA adducts of the food-derived mutagen 2-amino-9 H-pyrido-[2,3- b]indole (A( α)C) and bioassay of mammary gland carcinogenicity in Sprague–Dawley rats

2-amino-9 H-pyrido[2,3- b]indole (A αC) is a heterocyclic amine found at relatively high concentrations in barbecued or grilled meats. In the current study, the mammary gland carcinogenicity of A αC was examined in female Sprague–Dawley rats given 10 doses of A αC (75 mg/kg, orally, once per day starting at 43 days of age) and placed on a defined high-fat diet (23.5% corn oil), a strong promotional factor for rat mammary gland carcinogenesis. Within 1 year, one out of 20 rats dosed with A αC developed a tubulopapillary carcinoma, indicating that the bioassay was largely negative. As DNA adduct formation is considered to play a role in carcinogenesis, A αC–DNA adduct levels were measured in the mammary gland and other tissues by the 32P-postlabelling method. Under intensification conditions, one major adduct and up to three minor adducts were detected in isolated mammary gland epithelial cells and other tissues (liver, stomach, small intestine, colon and kidney) of A αC-treated rats; the adduct patterns were similar in all tissues examined. The major adduct, comprising 60–100% of total DNA adduct levels in tissues, was chromatographically identical to the principal adduct found in 3′-dGp-A αC (synthesized by reacting 3′-phospho-2′-deoxyguanosine (3′-dGp) with N-acetoxy-A αC). Of the tissues examined, the highest A αC–DNA adduct levels were found in the liver. In male rats given a single dose of A αC (75 mg/kg, orally, 3 hr prior to necropsy), no A αC–DNA adducts were detected in extrahepatic tissues. In female rats given a single dose or 12 daily doses of A αC, hepatic DNA adduct levels were at least 12–13-fold higher than those in any other tissue. Mean total A αC–DNA adduct levels in mammary gland epithelial cells and liver from female rats given multiple doses of A αC were 3.5 and 50.7 (RAL×10 7), respectively. Although factors in addition to DNA adduct formation are likely to play a role in mammary gland carcinogenesis, the results suggest that the weak mammary gland carcinogenicity of A αC may in part be associated with low A αC–DNA adduct levels in the mammary gland epithelium.

Molecular epizootiology of genotoxic events in marine fish: linking contaminant exposure, DNA damage, and tissue-level alterations.

Molecular epizootiological studies are increasingly being used to investigate environmental effects of genotoxic contaminants. The assessment of damage to DNA and linking the damage to subsequent molecular, cellular, or tissue-level alterations is a central component of such studies. Our research has focused on the refinement of the 32P-postlabeling assay for measuring covalent DNA-xenobiotic adducts arising from exposure to polycyclic aromatic compounds, using DNA adducts as molecular dosimeters of genotoxic contaminant exposure in biomonitoring studies, and investigating the relationship of DNA adduct formation to toxicopathic liver disease, including neoplastic lesions. A combination of field and laboratory studies using the 32P-postlabeling assay has shown that DNA adducts in marine fish are effective molecular dosimeters of genotoxic contaminant exposure. Investigations of the relationship of DNA adduct formation to neoplastic liver disease have shown that elevated levels of DNA adducts in certain fish species from contaminated coastal sites are associated with increased prevalences of toxicopathic hepatic lesions, including neoplasms, and that the ability to assess DNA damage has helped to explain, in part, species differences in lesion prevalence. Moreover, in a study of a site in Puget Sound contaminated with polycyclic aromatic compounds, we have shown, for the first time, that elevated levels of hepatic DNA adducts are a significant risk factor for certain degenerative and preneoplastic lesions occurring early in the histogenesis of hepatic neoplasms in feral English sole (Pleuronectes vetulus). These latter findings coupled with our current studies of mutational events in the K-ras proto-oncogene should provide further mechanistic substantiation that mutagenic events resulting from exposure to complex mixtures of genotoxic polycyclic aromatic compounds are involved in the etiology of hepatic neoplasia in English sole.

Determination of DNA damage in F344 rats induced by geometric isomers of tamoxifen and analogues.

To investigate the activation mechanisms involved in tamoxifen carcinogenicity, analogues of tamoxifen isomers modified at the ethyl group were synthesized and assessed for their ability to induce hepatic DNA damage following their administration to female F344 rats. The cis isomer was prepared by acid-catalyzed isomerization of tamoxifen and isolated by preparative HPLC. The active metabolite alpha-hydroxytamoxifen and geometric isomers of bromotamoxifen and C-desmethylenetamoxifen, analogues in which the ethyl group has been replaced by a bromine atom and methyl group, respectively, were synthesized according to published procedures. The levels of hepatic DNA adducts induced were determined by 32P-postlabeling. Bromotamoxifen and tamoxifen 1,2-epoxide caused no detectable DNA damage relative to controls. Trans isomers of tamoxifen, C-desmethylenetamoxifen, and alpha-hydroxytamoxifen all produced DNA adducts at a 5-90-fold higher level than the corresponding cis isomers. In contrast, both the cis and trans isomers of alpha-hydroxytamoxifen showed similar reactivity toward calf thymus DNA in vitro. Molecular models of alpha-hydroxytamoxifen isomers suggest this difference in DNA adduct-forming ability is due to steric hindrance of the enzymes involved in the activation of this metabolite. There were high adduct levels in the liver, but no uterine DNA adducts were detected in rats treated with alpha-hydroxytamoxifen. This suggests that in contrast to the liver, alpha-hydroxytamoxifen is not further activated in rat uterus. This may help to explain the absence of uterine tumors in rats following long-term tamoxifen treatment.

Accumulation and dose-response of hepatic DNA adducts in English sole ( Pleuronectes vetulus) exposed to a gradient of contaminated sediments

Levels of hepatic DNA adducts and concentrations of fluorescent aromatic compounds (FACs) in bile were measured in English sole ( Pleuronectes vetulus) exposed for up to 5 weeks to a reference sediment amended with a sediment containing high concentrations of polycyclic aromatic compounds (PACs) from Eagle Harbor, Puget Sound, WA. Levels of hepatic DNA adducts increased linearly with both concentration of sediment polycyclic aromatic hydrocarbons (PAHs) and length of exposure, whereas concentrations of biliary FACs were dose-responsive to levels of sediment PAHs but attained steady-state concentrations after 2 weeks of exposure. The levels of DNA adducts and concentrations of biliary FACs in fish exposed to the reference sediment remained at baseline levels throughout the exposure. Formation of PAC-DNA adducts was observed in liver of English sole injected with an extract of Eagle Harbor sediment (EHSE) and in English sole hepatocytes incubated with EHSE or the neutral fraction of EHSE containing predominantly PACs. In addition, DNA adduct and biliary FAC levels measured in feral English sole captured from Eagle Harbor, when compared with levels measured in fish from the laboratory sediment exposure study, also suggested accumulation of DNA adducts in the fish captured from Eagle Harbor. These findings, in conjunction with previous studies showing PAC-DNA adducts are persistent in fish, suggest that a substantial proportion of PAC-induced DNA damage was not readily repaired in English sole, and thus, measurement of hepatic DNA adducts can be used as an indicator of cumulative exposure to genotoxic PACs.

In vivo mutagenicity and DNA adduct levels of heterocyclic amines in Muta ™Mice and [formula omitted] double transgenic mice

The cooked meat derived heterocyclic amines (HCAs) 2-amino-3-methylimidazo[4,5- f]quinoline (IQ), 2-amino-3,8-dimethylimidazo[4,5- f]quinoxaline (MeIQx), and 2-amino-9 H-pyrido[2,3- b]indole (AαC) are established mutagens in the Salmonella assay and hepatocarcinogens in mice. The current study uses transgenic mice to examine hepatic HCA-DNA adduct formation and mutagenesis in vivo and the impact of hepatic overexpression of the c- myc oncogene on HCA-induced mutagenesis. C57Bl lacZ and c-myc lacZ mice strains, produced by crossbreeding Muta ™Mice (carrying the lacZ mutation target gene) with either C57Bl control or c- myc transgenic mice, respectively, were treated with 10 daily doses of IQ, MeIQx or AαC (20 μg/g, p.o.). Four weeks after dosing, the frequency of mutations in the lacZ gene in liver of either C57Bl lacZ or c-myc lacZ mice was significantly higher in mice treated with any one of the three HCAs than in mice given vehicle only. In addition, all three HCAs formed hepatic DNA adducts, as measured by the 32P-postlabeling analysis 24 h after dosing. In both strains of mice, hepatic DNA adduct levels were 2–3-fold higher with AαC than with either IQ or MeIQx, although the mutant frequencies in the lacZ gene were 30–40% lower in mice dosed with AαC. These results suggest that AαC-DNA adducts may be less mutagenic in vivo than either IQ- or MeIQx-DNA adducts. The lacZ mutant frequencies observed with all three HCAs appeared to be influenced by c- myc transgene expression: after HCA treatment, transgenic mice carrying the c- myc gene showed a 30–40% higher lacZ mutant frequency than mice not carrying this transgene. Notably, lacZ mutant frequencies were not different among C57Bl lacZ and c-myc lacZ mice that received vehicle control. DNA adduct studies showed that the levels of IQ- and MeIQx-DNA adducts were 2–3-fold higher in c-myc lacZ mice than in C57Bl lacZ mice; however, AαC-DNA adducts were not statistically different between the two strains. In addition, phase I metabolic activation of these HCAs, as assessed by hepatic microsomal mutagenic activation, was also similar in both strains of mice. These results support the notion that overexpression of the c- myc oncogene cooperates with the HCAs to enhance in vivo mutagenicity. Further studies are needed to assess the mechanisms of this cooperative effect.

Chemical Contaminant Exposure and Effects in Four Fish Species from Tampa Bay, Florida

Concentrations of selected anthropogenic chemical contaminants and levels of pollution-related biological effects were measured during three consecutive years (1990–1992) in hardhead catfish (Arius felis), Gulf killifish (Fundulus grandis), longnose killifish (F. majalis), and red drum (Scieaenops ocellatus) from 12 subtidal and intertidal sites in Tampa Bay and nearby Sarasota Bay. Each species was collected from at least four sites. Compared to nonindustrialized sites, concentrations of PCBs, DDTs, and alpha-chlordane in liver, and of fluorescent aromatic compounds in bile, were highest in fish from sites in or near Hillsborough Bay, the most industrialized portion of Tampa Bay. The results of analyses for two biochemical markers of contaminant-induced effects in fish, hepatic cytochrome P4501A activities and levels of hepatic DNA adducts, also showed the highest levels to be in all four fish species from sites in the vicinity of Hillsborough Bay. Liver lesions, considered to be pollution-associated in several other bottom-feeding fish species, were found in hardhead catfish and longnose killifish, exclusively from sites in Hillsborough Bay. Overall, concentrations of selected contaminants and their derivatives in the four target fish species generally reflected concentrations of these contaminants found in sediment. The biochemical and histopathological responses demonstrated that chemical contaminant concentrations in the vicinity of Hillsborough Bay are sufficiently high to cause adverse effects in indigenous fish species. The results, collectively, showed that the extent of contaminant exposure and biological effects in fish from sites in Tampa Bay were low to moderate compared to more urbanized coastal sites of the United States. *** DIRECT SUPPORT *** A01BY073 00009

Contaminant exposure and biochemical effects in outmigrant juvenile chinook salmon from urban and nonurban estuaries of puget sound, Washington

AbstractJuvenile chinook salmon (Oncorhynchus tshawytscha) were sampled in Puget Sound, Washington, for 2 consecutive years from contaminated urban estuaries, a nonurban estuary, and from the respective hatcheries to assess exposure to anthropogenic chemicals and to determine if biochemical changes were occurring as a consequence of exposure. Exposure to polycyclic aromatic hydrocarbons (PAHs), chlorinated hydrocarbons, and butyltins was determined. The mean concentrations of PAHs and PCBs in stomach contents and PCBs in liver were significantly higher in salmon from the urban estuaries compared to fish from the nonurban estuary in both sampling years. Higher hepatic concentrations of PCBs than DDTs were found in fish from the urban estuaries, but butyltins were rarely detected. Further, mean concentrations of fluorescent aromatic compounds in bile, an estimate of exposure to PAHs, and hepatic cytochrome P4501A and levels of hepatic DNA adducts were also significantly higher in salmon from the urban estuaries compared to either the nonurban estuary or the hatcheries. Results demonstrated increased exposure to chemical contaminants in outmigrant juvenile salmon during their relatively brief residence in urban estuaries of Puget Sound. Moreover, the exposure was sufficient to elicit biochemical responses, which suggest a potential for other biological effects to ensue.

CONTAMINANT EXPOSURE AND BIOCHEMICAL EFFECTS IN OUTMIGRANT JUVENILE CHINOOK SALMON FROM URBAN AND NONURBAN ESTUARIES OF PUGET SOUND, WASHINGTON

Juvenile chinook salmon (Oncorhynchus tshawytscha) were sampled in Puget Sound, Washington, for 2 consecutive years from contaminated urban estuaries, a nonurban estuary, and from the respective hatcheries to assess exposure to anthropogenic chemicals and to determine if biochemical changes were occurring as a consequence of exposure. Exposure to polycyclic aromatic hydrocarbons (PAHs), chlorinated hydrocarbons, and butyltins was determined. The mean concentrations of PAHs and PCBs in stomach contents and PCBs in liver were significantly higher in salmon from the urban estuaries compared to fish from the nonurban estuary in both sampling years. Higher hepatic concentrations of PCBs than DDTs were found in fish from the urban estuaries, but butyltins were rarely detected. Further, mean concentrations of fluorescent aromatic compounds in bile, an estimate of exposure to PAHs, and hepatic cytochrome P4501A and levels of hepatic DNA adducts were also significantly higher in salmon from the urban estuaries compared to either the nonurban estuary or the hatcheries. Results demonstrated increased exposure to chemical contaminants in outmigrant juvenile salmon during their relatively brief residence in urban estuaries of Puget Sound. Moreover, the exposure was sufficient to elicit biochemical responses, which suggest a potential for other biological effects to ensue.

Accumulation and persistence of DNA adducts of the synthetic steroid cyproterone acetate in rat liver.

Cyproterone acetate (CPA) is a synthetic steroid which is widely used in antiandrogenic and gestagenic drugs. We have recently shown that CPA induces DNA adducts in cultured rat hepatocytes and in rat liver (1). In the present investigation, we studied the persistence and accumulation of CPA-derived DNA adducts in the liver of rats using the 32P-postlabeling technique. To study the persistence of CPA-DNA adducts, rats were treated with a single oral dose of 10 (female rats) or 100 mg CPA/kg body wt (male rats). Four DNA adducts were detected in the liver of both gender. In female rats, maximal total DNA adduct levels of 3.40 +/- 0.04 adducts/10(6) nucleotides were observed after 1 week. Eleven weeks later, 40% of the adducts determined after 1 week were still detectable. In male rats, maximal hepatic DNA adduct levels of approximately 98 +/- 3/10(9) nucleotides were attained after 2 weeks. The adduct level decreased during the following 4 weeks to approximately 40% of the earlier observed maximal level. To study the accumulation of the CPA-DNA adducts, rats were treated daily with a low oral dose of 50 micrograms CPA/kg body wt for 42 days. During this treatment period, the level of the four adducts increased continuously from approximately 10 to approximately 380 adducts/10(9) nucleotides in the liver of female rats. DNA adducts were formed at much lower levels in male rats; only one type of DNA adduct was detectable, the level of which increased to approximately 6 adducts/10(9) nucleotides after 42 days. In conclusion, CPA induces DNA adducts in rat liver; binding of the steroid is much higher in female compared to male rats. The CPA-DNA adducts show a high persistence and as a consequence of their long half life, CPA-DNA adducts accumulate significantly in the liver of rats.

A Biomarker Approach to Assessing Xenobiotic Exposure in Atlantic Tomcod from the North American Atlantic Coast

We determined levels of hepatic cytochrome P4501A (CYP1A) mRNA, hepatic DNA adducts, and fluorescent aromatic compounds (FACs) in bile, a measure of exposure to polyaromatic hydrocarbons, in Atlantic tomcod from six river systems ranging from highly polluted to relatively pristine on the northeast North American coast (the Hudson River, New York; the St. Lawrence River, Quebec; the Miramichi River, New Brunswick; the Saco and Royal rivers, Maine; and the Margaree River, Nova Scotia). Hudson River tomcod showed the greatest response for all parameters, and tomcod from the Margaree River exhibited the least response. Tomcod from the Miramichi River exhibited marked induction of CYP1A mRNA but low levels of hepatic DNA adducts and biliary FACs, whereas fish from the St. Lawrence River showed no induction of CYP1A mRNA and moderately elevated levels of DNA adducts and biliary FACs. In tomcod from the Hudson and Miramichi rivers, the levels of CYP1A mRNA were 28 times and 14 times, respectively, as great as the levels in fish from the St. Lawrence, Saco/Royal, and Margaree rivers. Mean levels of DNA adducts varied from 120 nmol adducts/mol bases in Hudson River tomcod to &lt; 3 nmol adducts/mol bases in fish from the Miramichi and Margaree rivers. Concentrations of FACs in the bile of tomcod from the Hudson and St. Lawrence rivers were 8 and 1.8 times, respectively, as great as the concentrations in tomcod from the Miramichi River and Margaree River. In tomcod from the Hudson River, all three biomarkers were markedly elevated; in the St. Lawrence River two biomarkers were elevated, in the Miramichi River one was elevated, but no biomarker was substantially elevated in fish from the Saco/Royal and Margaree rivers. Elevated levels of hepatic DNA adducts and biliary FACs in tomcod from the Hudson River suggest increased exposure to PAHs, consistent with previous studies.

Biochemical markers in feral roach ( Rutilus rutilus) in relation to the bioaccumulation of organic trace pollutants

The levels of organic trace contaminants, including polyaromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs) and organochlorine pesticides (OCPs), were analyzed in sediments and in roach ( Rutilus rutilus, a common fish in Dutch freshwater) from two Amsterdam lakes with different pollutant levels. Roach muscle tissue levels of PCBs and OCPs reflected those found in the sediments. PAH muscle tissue levels, however, were higher in roach from the less polluted lake. In addition, a suite of biochemical parameters was measured in roach livers in order to evaluate their utility as biomarkers of aquatic pollution. None of the phase I-enzymes (total cytochrome P450 [cyt P450], cytochrome b 5 [cyt b 5], cytochrome P450 1A [P450 1A], ethoxyresorufin O-deethylase [EROD] and NADPH cytochrome c reductase [RED]) appeared to be induced in roach from the polluted site. On the contrary, cyt P450 and cyt b 5 were significantly inhibited in roach from the more polluted lake. These findings were consistent with the results of measurements on phase II-enzymes. Glutathione S-transferase [GST] activity was significantly inhibited in roach from the polluted site, while no significant changes in the co-factor levels of glutathione [GSH or GSSG] were observed. Hepatic DNA adduct levels were the same in roach from the more polluted and the less polluted lake. Accordingly, the hepatic biochemical parameters measured in this study cannot be considered reliable indicators of chemical pollution or contaminant stress in roach from the polluted lake.

Biphasic Removal of DNA Adducts in a Repetitive DNA Sequence after Dietary Administration of 2-Acetylaminofluorene

Dietary administration of the hepatocarcinogen 2-acetylaminofluorene (2-AAF) to rats results in the formation of a major hepatic DNA adduct, N-(deoxyguanosin-8-yl)-2-aminofluorene (dG-C8-AF). In liver DNA, dG-C8-AF reaches steady-state conditions after approximately 2 weeks of feeding and is removed in a biphasic manner. In these experiments, we have quantified adduct concentrations in a 370 base-pair repetitive DNA fragment to determine if the adduct levels and kinetics of adduct removal were similar to those found in total genomic DNA. Male F344 rats were fed 0.02% 2-AAF for 28 days and were sacrificed at intermittent times up to 56 days after being returned to the control diet. Hepatic DNA adduct levels were measured by 32P-postlabeling or radioimmunoassay (RIA) in total genomic DNA and in a 370 base-pair fragment obtained by digesting genomic DNA with Hind III. Biphasic removal of dG-C8-AF, which composed about 90% of the total adducts measured, was observed in total genomic DNA, with comparable rate constants being detected by both 32P-postlabeling and RIA. 32P-Postlabeling also showed analogous biphasic removal of dG-C8-AF in the 370 base-pair fragment. A second adduct, 3-(deoxyguanosin-N2-yl)-2-AAF (dG-N2-AAF), which accounted for about 10% of the total adducts measured, showed similar biphasic removal kinetics in the total genomic DNA and the 370 base-pair fragment; however, as compared to dG-C8-AF, little removal of dG-N2-AAF was observed during the slow phase.