The metabolism of benzo(a)pyrene by English sole (Parophrys vetulus): comparison between isolated hepatocytes in vitro and liver in vivo.

1. Metabolites and DNA adducts of 3H-benzo(a)pyrene (BaP) formed by isolated hepatocytes from English sole (Parophrys vetulus) in vitro were compared to those in bile and liver of sole exposed i.m. to 3H-BaP. 2. English sole liver was perfused with a collagenase solution and hepatocytes were isolated with greater than 95% viability. Determination of kinetic parameters for metabolism of 3H-BaP showed a Km of 29 +/- 10 microM and an apparent Vmax of 1300 pmol BaP metabolized/10(6) cells per h. 3. Analysis of medium from hepatocyte cultures and bile by ion-pair h.p.l.c. showed significant amounts of radioactivity in regions where glucuronide and glutathione conjugates of BaP metabolites elute. No sulphate conjugates of BaP metabolites were detected. The major unconjugated metabolite formed by hepatocytes was the BaP-9,10-dihydrodiol. 4. Hydrolysis of glucuronide conjugates by beta-glucuronidase and reversed-phase h.p.l.c. analysis of chloroform-soluble metabolites showed the presence of BaP-7,8-dihydrodiol, 1-hydroxyBaP and 3-hydroxyBaP. The identities of these metabolites were confirmed by comparing their fluorescence spectra with those of standard BaP metabolites. 5. Analysis by 32P-postlabelling of the BaP-DNA adducts formed in isolated hepatocytes and liver revealed that major adducts detected are derived from the anti-7,8-dihydrodiol-9,10-epoxideBaP (anti-BaPDE) and syn-BaPDE. 6. Results show that the types of conjugated metabolites and BaP-DNA adducts formed in primary hepatocyte culture were similar to those in bile and liver of English sole exposed to BaP. Thus, isolated hepatocytes from English sole afford a reliable alternative to live fish for studies of the mechanisms of hepatic xenobiotic metabolism and DNA adduct formation in a species shown to be susceptible to induction of hepatocarcinogenesis by PAHs.     

DNA adduct measurements in zebra mussels, Dreissena polymorpha, Pallas. Potential use for genotoxicant biomonitoring of fresh water ecosystems

The purpose of this study was to examine PAH accumulation and bulky DNA adduct formation in the digestive gland of zebra mussels exposed in their habitat or in controlled laboratory conditions to complex mixture of PAH. DNA adducts were measured using a 32P-postlabelling protocol with nuclease P1 enrichment adapted from Reddy and Randerath [Reddy, M.V., Randerath, K., 1986. Nuclease P1-mediated enhancement of sensitivity of 32P-postlabelling test for structurally diverse DNA adducts. Carcinogenesis 7, 1543-1551]. Specimens collected in the upper part of the Seine estuary were shown to accumulate higher levels of PAH (up to 1.6 microg g(-1) dry weight) in comparison to individuals from the reference site (0.053 microg g(-1) dry weight). The former exhibited elevated levels of DNA adducts (up to 4.0/10(8) nucleotides) and higher diversity of individual adducts with five distinct spots being specifically detected in individuals originating from the Seine estuary. Zebra mussels exposed for 5 days to 0.01% (v/v) of organic extract of sediment from the Seine estuary were shown to accumulate high amounts of PAH (up to 138 microg g(-1) dry weight) but exhibited relatively low levels of DNA adducts. Exposure to benzo[a]pyrene led to a dose-dependent accumulation of B[a]P (up to 7063 microg g(-1) dry weight) and a clear induction of DNA adduct formation in the digestive gland of mussels (up to 1.13/10(8) nucleotides). Comparisons with other bivalves exposed to the same model PAH, revealed similar levels of adducts and comparable adduct profiles with a main adduct spot and a second faint one. This study clearly demonstrated that zebra mussels are able to biotransform B[a]P and probably other PAH into reactive metabolites with DNA-binding activity. This work also demonstrated the applicability of the nuclease P1 enhanced 32P-postlabelling method for bulky adduct detection in the digestive gland of zebra mussels. DNA adduct measurement in zebra mussels could be a suitable biomarker to monitor PAH-exposure and evaluate genotoxicity in fresh water ecosystems.     

Persistence of benzo[a]pyrene--DNA adducts in hematopoietic tissues and blood of the mummichog, Fundulus heteroclitus

The formation and persistence of benzo[a]pyrene (B[a]P)-DNA adducts were investigated in blood, liver and two hematopoietic tissues (anterior kidney and spleen) of the mummichog (Fundulus heteroclitus). Fish were injected with a single, sublethal dose of B[a]P (12 mg/kg body weight) and sampled from 8 to 96 days post-injection. 32P-Postlabeling analysis and storage phosphor imaging were used to resolve and quantify hydrophobic DNA adducts. One major DNA adduct was present in each of the examined tissues at all sampling times. This adduct had similar chromatographic characteristics to those of the adduct standard, 7R,8S,9S-trihydroxy-10S-(N(2)-deoxyguanosyl-3'-phosphate)-7,8,9,10-tetrahydro-benzo[a]pyrene (B[a]PDE-dG). Minor DNA adduct spots, representing less than 2% of the total DNA adducts, were observed in some liver, anterior kidney and spleen samples for up to 32 days post-injection. The B[a]P-DNA adducts reached maximal levels at 32 days post-injection and persisted for at least 96 days in all examined tissues. B[a]P-DNA adduct levels were significantly higher in the liver and anterior kidney than in the spleen from 16 to 96 days (P<0.001), although liver and anterior kidney DNA adduct levels were not significantly different at any time. This is the first controlled study to demonstrate the formation and persistence of B[a]P-DNA adducts in hematopoietic tissues and blood of fishes exposed to the prototypical polycyclic aromatic hydrocarbon, B[a]P. Although persistent DNA adducts are generally recognized as potential initiators of carcinogenic processes, adducts in these vital tissues may also lead to disruption of physiological functions such defense mechanisms and hematopoiesis.     

Effects of adsorption of benzo[a]pyrene onto carbon black particles on levels of DNA adducts in lungs of rats exposed by inhalation

Exposure of rodents to benzo[a]pyrene (BaP) associated with particles has previously been shown to result in increased retention of BaP and metabolites in lungs. To determine if DNA damage might be enhanced, DNA adducts were measured in lungs of F344 rats following inhalation of pure BaP aerosols or BaP absorbed on carbon black particles. Groups of rats were exposed nose only to filtered air, [14C]BaP (2 mg/m3), or [14C]BaP (2 mg/m3) adsorbed on carbon black (97 mg/m3) (BaP/CB) for 4 hr/day, 1 day/week, for 12 weeks. Groups of rats were terminated at 4, 8, 12, 16, 20, and 24 weeks after the beginning of the 12-week exposure period. Retention of total 14C in lungs was used as an indicator of total reactive metabolites. DNA isolated from lungs was analyzed for adducts using a 32P-postlabeling assay. Inhalation of BaP/CB resulted in 100-fold higher levels of 14C in lungs at the end of the 12-week exposure than did inhalation of pure BaP. The halftime for the decline in 14C levels was 34 +/- 3 weeks (mean +/- SE) for rats exposed to BaP/CB and 6 +/- 2 weeks for rats exposed to pure BaP. At the end of 12 weeks of exposure, DNA adducts in lungs of rats exposed to pure BaP ranged from 2-15 adducts per 10(9) bases (mean = 7, n = 4) and in rats exposed to pure BaP absorbed on carbon black ranged from 10-12 adducts per 10(9) bases (mean = 11, n = 4); DNA adducts in lungs of sham-exposed rats ranged from 0-2 adducts per 10(9) bases (mean = 1, n = 4). The halftimes for the decline in DNA adducts in lungs were 3 +/- 1 weeks (mean +/- SE) for the rats exposed to BaP/CB and 5 +/- 2 weeks for the rats exposed to BaP. One of the DNA adducts found following exposure to both BaP and BaP/CB was tentatively identified as the BaP diol epoxide deoxyguanosine (BPDE) adduct. Levels of both total and BPDE DNA adducts were significantly increased (p less than 0.05) in lungs of rats exposed to both BaP and BaP/CB compared to levels in lungs of sham-exposed rats. There were no significant differences in levels of DNA adducts in lungs of rats exposed to BaP or BaP/CB, although the pattern of adducts was different between the two exposure modes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ovarian susceptibility to benzo[a]pyrene: tissue burden of metabolites and DNA adducts in F-344 rats

Exposure to environmental toxicants has been implicated as one of the causative factors for infertility in mammals. The objective of this study was to determine the amount of ingested benzo[a]pyrene (BaP), an environmental toxicant that reaches the reproductive tissues (internal dose) subsequent to a single acute exposure. Toward this end, the concentrations of BaP reactive metabolites and BaP-DNA adducts were measured throughout the course of BaP's residence in the body. Ten-week-old female Fischer-344 rats weighing approximately 220 g were administered 5 mg BaP/kg body weight orally. 1, 7, 14, 2,1 and 28 d post BaP exposure, BaP parent compound and metabolites from plasma, ovaries, and liver tissues were extracted using liquid-liquid extraction. The extracts were analyzed by reverse-phase highperformance liquid chromatography (HPLC). DNA was isolated and analyzed for BaP-induced DNA adducts by (32)P-postlabeling method. The BaP total metabolite concentrations in plasma, ovaries, and liver showed a gradual decrease from d 1 to 28 post BaP administration. The BaP-DNA adducts concentrations in ovaries and liver tissues from the treatment group demonstrated a trend similar to that observed for metabolites. Ovaries showed greater concentrations of DNA adducts compared to liver. However, with an increase in time post cessation of exposure, the adduct concentrations in liver tissue started declining rapidly, from d 1 to 28. For ovaries, the adduct concentrations demonstrated a significant decline from d 1 to 7 and a gradual fall thereafter. A concordance between BaP reactive metabolite levels and adduct concentrations indicates that the bioavailability of reactive metabolites determines the binding with DNA and consequently the formation and persistence of adducts in an acute exposure regimen.     

DNA adduct formation in precision-cut rat liver and lung slices exposed to benzo[a]pyrene.

Chemical-DNA adducts provide an integrated measure of exposure, absorption, bioactivation, detoxification, and DNA repair following exposure to a genotoxic agent. Benzo[a]pyrene (BaP), a prototypical polycyclic aromatic hydrocarbon (PAH), can be bioactivated by cytochrome P-450s (CYPs) and epoxide hydrolase to genotoxic metabolites which form covalent adducts with DNA. In this study, we utilized precision-cut rat liver and lung slices exposed to BaP to investigate tissue-specific differences in chemical absorption and formation of DNA adducts. To investigate the contribution of bioactivating CYPs (such as CYP1A1 and CYP1B1) on the formation of BaP-DNA adducts, animals were also pretreated in vivo with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) prior to in vitro incubation of tissue slices with BaP. Furthermore, the tissue distribution of BaP and BaP-DNA adduct levels from in vivo studies were compared with those from the in vitro tissue slice experiments. The results indicate a time- and concentration-dependent increase in tissue-associated BaP following exposure of rat liver and lung tissue slices to BaP in vitro, with generally higher levels of BaP retained in lung tissue. Furthermore, rat liver and lung slices metabolized BaP to reactive intermediates that formed covalent adducts with DNA. Total BaP-DNA adducts increased with concentration and incubation time. Adduct levels (fmol adduct/microg DNA) in lung slices were greater than liver at all doses. Liver slices contained one major and two minor adducts, while lung slices contained two major and 3 minor adducts. The tissue-specific qualitative profile of these adducts in tissue slices was similar to that observed from in vivo studies, further validating the use of this model. Pretreatment of animals with TCDD prior to in vitro incubation with BaP potentiated the levels of DNA adduct formation. TCDD pretreatment altered the adduct distribution in lung but not in liver slices. Together, the results suggest that tissue-specific qualitative and quantitative differences in BaP-DNA adducts could contribute to the lung being a target tissue for BaP carcinogenesis. Furthermore, the results validate the use of precision-cut tissue slices incubated in dynamic organ culture as a useful model for the study of chemical-DNA adduct formation.     

Dosimetry by means of DNA and hemoglobin adducts in propylene oxide-exposed rats

The main purpose of the study was to establish the relation between exposure dose of propylene oxide (PO) and dose in various tissues of male F344 rats exposed to the compound by inhalation. The animals were exposed to 0, 5, 25, 50, 300, or 500 ppm PO in the air for 3 days (6 h/day) or 4 weeks (6 h/day, 5 days/week). Blood, nasal respiratory epithelium, lung, and liver were collected. 2-Hydroxypropylvaline (HPVal) in hemoglobin was quantified using the N-alkyl Edman method and gas chromatography/tandem mass spectrometry. 7-(2-Hydroxypropyl)guanine (7-HPG) in DNA was quantified using (32)P postlabeling. The levels of 7-HPG in DNA of nasal respiratory epithelium and lung increased linearly with concentration as measured both after 3 days and 4 weeks of exposure. Similarly, 7-HPG in liver DNA and HPVal in hemoglobin showed a linear increase with PO concentration in the 3-day exposure group, whereas a deviation from linearity was observed above 300 ppm in the 4-week exposure group. The new results confirm previous observations of a dose difference between tissues with the highest dose present in the nasal respiratory epithelium. The measured adduct levels were used for calculation of adduct increments and corresponding tissue doses per unit of external exposure dose. For this purpose, the buildup of adducts was modeled considering the different kinetics of formation and elimination of adducts with DNA and hemoglobin, respectively, and also considering the increasing body weight of the animals. The half-life of 7-HPG in vivo, as well as tissue doses, could be solved from DNA adduct data at the 3rd and 26th days. Within the range of concentrations where the dose-response curves for adduct formation are linear, the relationship between exposure dose and resulting tissue doses could be based equally well on adduct data from the short-term exposure as on adduct data from the prolonged exposure.     

Molecular dosimetry of DNA adducts in rainbow trout (Oncorhynchus mykiss) exposed to benzo(a)pyrene by different routes

 Farm raised rainbow trout (Oncorhynchus mykiss) were exposed by various routes to benzo(a)pyrene (BP) as a representative carcinogenic polycyclic aromatic hydrocarbon (PAH). Following exposure of fish to the chemical by intraperitoneal (i.p.) injection, ³²P-postlabelling studies indicated that non-feral trout were relatively resistant to the formation of BP-DNA adducts in liver. No adducts were detected in fish exposed to single doses (20 mg/kg) of BP. Multiple exposures (e.g. 2×25 mg/kg) were necessary in order for adducts to be detected, indicating that induction of the metabolising enzymes required for the bioactivation of BP is necessary. These studies provided reference information on DNA adducts for comparison with data from subsequent experiments at environmentally realistic low level exposures. Two types of low level aquatic exposure were carried out. The first procedure exposed fish for 30 days to a nominally constant low level (1.2 and 0.4 μg/l) of a homogeneous dispersion of BP in water, to simulate low level aquatic environmental exposures. Following ³²P-postlabelling analysis of the liver DNA of exposed fish, BP-DNA adducts were not detected. In the second procedure, fish were exposed to a constant low level of BP (ca. 0.5 μg/l) for 15 days then to a pulse (60 μg/l) which was allowed to naturally decline (to ca. 2 μg/l) during a further 15 days. Following this exposure, significant levels of BP-DNA adducts were detected in livers of trout. The effect of dietary exposures was investigated by feeding trout a diet containing either 58 μg or 288 μg BP per day for 6 days, equivalent to total doses of 43 mg/kg and 216 mg/kg. In both cases BP-DNA adducts were detected in livers of exposed fish. The results provide useful information on the types of exposures to PAHs which may pose a genotoxic risk to fish in the environment. Received: 5 April 1994 / Accepted: 31 May 1994     

Benzo[a]pyrene-induced immunotoxicity: comparison to DNA adduct formation in vivo, in cultured splenocytes, and in microsomal systems

Benzo[a]pyrene (BaP)/DNA adduct formation appears to be involved in carcinogenesis, but the relationship between adduct formation and BaP-induced immunotoxicity is unknown. We compared DNA adduct formation (32P-postlabeling analysis) to suppression of polyclonal immune responses (3H-TdR incorporation and IgM secretion) and decreases in cell viability in B6C3F1 female mouse splenic leukocytes (SPL). BaP administration (200 mg/kg, ip) resulted in suppression of polyclonal responses and substantial DNA adduct formation in mouse SPL. SPL adduct levels were similar to those in liver, lung, kidney, and stomach. In vitro exposure of SPL to BaP without rat liver activation enzymes (S9) caused decreases in SPL viability and immune responses that were dependent on dose and exposure period. However, DNA adduct formation in SPL was very low between 1 and 200 microM BaP. S9 enhanced the toxicity of BaP for SPL cultures. Adduct formation was rapid and dose related in +S9 incubates. The low level of BaP activation by SPL was confirmed in microsomal incubations in which splenic microsomes exhibited much lower aryl hydrocarbon hydroxylase (AAH) activity and ability to form DNA-adducting metabolites than did microsomes from liver or lung. Results indicate that immunosuppression produced by BaP in these systems was due to cytotoxic effects. It appears that these effects were caused by two separate mechanisms, one dependent on and one independent of DNA adduct formation. Since SPL had high levels of DNA adducts after ip injection of BaP, reactive metabolites of BaP may be involved in the immunotoxicity seen in vivo.     

Potential Application of Benzo(a)Pyrene-Associated Adducts (Globin or Lipid) as Blood Biomarkers for Target Organ Exposure and Human Risk Assessment

In order to investigate the potential application of blood biomarkers as surrogate indicators of carcinogen–adduct formation in target-specific tissues, temporal formation of benzo[a]pyrene (BaP)-associated DNA adducts, protein adducts, or lipid damage in target tissues such as lung, liver, and kidney was compared with globin adduct formation or plasma lipid damage in blood after continuous intraperitoneal (ip) injection of [³H]BaP into female ICR mice for 7 d. Following treatment with [³H]BaP, formation of [³H]BaP–DNA or –protein adducts in lung, liver, and kidney increased linearly, and persisted thereafter. This finding was similar to the observed effects on globin adduct formation and plasma lipid damage in blood. The lungs contained a higher level of DNA adducts than liver or kidneys during the treatment period. Further, the rate of cumulative adduct formation in lung was markedly greater than that in liver. Treatment with a single dose of [³H]BaP indicated that BaP–globin adduct formation and BaP–lipid damage in blood reached a peak 48 h after treatment. Overall, globin adduct formation and lipid damage in blood were significantly correlated with DNA adduct formation in the target tissues. These data suggest that peripheral blood biomarkers, such as BaP–globin adduct formation or BaP–lipid damage, may be useful for prediction of target tissue-specific DNA adduct formation, and for risk assessment after exposure.     

Development of a 32P-postlabelling method for the detection of 1,N 2-propanodeoxyguanosine adducts of crotonaldehyde in vivo

Crotonaldehyde is a genotoxic, mutagenic and carcinogenic alpha,beta-unsaturated carbonyl compound which forms 1,N2-propanodeoxyguanosine adducts. Humans are exposed to this compound at work places, and from tobacco smoke and air pollution, but also from food and beverages. Therefore crotonaldehyde can play a significant role in carcinogenesis. Since in vivo measurement of DNA adducts of crotonaldehyde can improve cancer risk assessment and contribute to the clarification of the role of crotonaldehyde in carcinogenicity, we developed, adapted and optimized a 32P-postlabelling technique for the adducts of crotonaldehyde based on nuclease P1 enrichment and on a polyethylene imine modified cellulose TLC to provide a detection sensitivity of three adducts per 10(9) nucleotides and a labelling efficiency of 80-90%. We also report a readily performable synthesis of adduct standards and demonstrated that DNA is completely digested to the 3'-monophosphate nucleotides under the conditions of our enzymatic DNA hydrolysis. We showed that the postlabelling method developed is appropriate for in vivo DNA-binding studies. Female Fischer 344 rats were treated by gavage with crotonaldehyde at doses of 200 and 300 mg/kg body weight, and 20 h after treatment adduct levels of 2.9 and 3.4 adducts per 10(8) nucleotides, respectively, were found in the liver DNA. Only 1.6 nucleotides per 10(8) nucleotides were found 12 h after treatment at 200 mg/kg body weight. Absolutely no adducts could be found in liver DNA of untreated rats with our method at the detection limit of three adducts per 10(9) nucleotides. In contrast to our group, the group of Chung have reported crotonaldehyde adduct levels in the range of 2.2 22 adducts per 10(8) nucleotides in DNA of untreated Fischer 344 rats. The clarification of this discrepancy is of importance for the elucidation of the role of crotonaldehyde in carcinogenicity, and both groups have decided to clarify this in cooperation in the near future.     

Postlabeling analysis of polycyclic aromatic hydrocarbon-DNA adducts in white blood cells of foundry workers

Blood samples were obtained from 61 volunteers working in a Finnish iron foundry who were exposed to carcinogenic polycyclic aromatic hydrocarbons and from 19 control subjects not known to be exposed to these chemicals. Foundry workers were categorized into high, medium, or low exposure groups, based on their exposure levels to airborne benzo[a]pyrene (BaP) (high greater than 0.2, medium 0.05-0.2, low less than 0.05 micrograms BaP/m3 air). Aromatic adducts were determined in white blood cell DNA from exposed and unexposed subjects using 32P-postlabeling. There was a highly significant correlation between the estimated exposure and adduct levels as determined by analysis of variance. The levels of adducts found in the high and medium group samples ranged from 5 to over 20 adducts in 10(8) DNA nucleotides. No effects due to age, sex, or the smoking habits of the subjects were observed. Within each group, the SEM appeared rather small. This study demonstrates the utility of the 32P-postlabeling assay to assess human exposure to known and unknown environmental aromatic genotoxicants.     

PAH metabolites in bile, cytochrome P4501A and DNA adducts as environmental risk parameters for chronic oil exposure: a laboratory experiment with Atlantic cod

In order to perform environmental risk assessments with regard to oil contamination in the sea, it is important to obtain knowledge about threshold levels for possible adverse effects in marine organisms. With this objective in mind, selected biomarkers were studied in Atlantic cod (Gadus morhua) chronically exposed to mechanically dispersed crude oil. The fish were exposed for 30 days in a continuous flow system to nominal concentrations of 0.06, 0.25 and 1 ppm North Sea crude oil. Fish were sampled five times during the exposure period. In addition, the 1 ppm group and the control group were sampled 1 week after the end of exposure. Polyaromatic hydrocarbon (PAH) concentrations in the seawater were analysed regularly by direct fluorescence and, at one occasion, by gas chromatography with mass spectrographic detection (GC/MS) measurements. Liver samples were analysed for parent PAH levels by means of GC/MS measurements, and PAH metabolites in bile were analysed by means of fixed wavelength fluorescence. Cytochrome P450 induction in liver was estimated by ethoxyresorufin-O-deethylase (EROD) activity, and hepatic DNA adducts were analysed by the 32P-postlabelling assay. The parent PAH concentrations in liver showed peak levels 3 days after the start of exposure, followed by a reduction towards the end of the experiment. In contrast, the PAH metabolites in bile and EROD activity showed generally increasing levels throughout the whole exposure period, indicating an increased biotransformation efficiency. The level of DNA adducts in the 1 ppm group showed a stable increase during the entire exposure period. Only a slight, non-significant decrease in DNA adduct levels was observed after 7 days of recovery in clean water. Exposure-dependent responses were observed for all three biomarkers. The lowest nominal concentration of dispersed oil in water, 0.06 ppm, corresponded to a measured total PAH concentration in the water of 0.3 ppb. Atlantic cod exposed to this concentration showed increased levels of PAH metabolites in bile and a slight induction of CYP1A, as well as formation of DNA adducts when compared with control fish. Particularly noteworthy is the detection of DNA adducts at such a low exposure concentration of oil in water, which, to our knowledge, is a novel finding. These dose-response data may serve as useful contributions when assessing environmental risk with regard to marine oil pollution.     

DNA adducts in carp exposed to artificial diesel-2 oil slicks

In attempts to mimic field exposure, oil slicks prepared from diesel-2 oil/water emulsions were poured onto the surface of water in tanks prepared fresh every day and liver DNA adducts were analyzed by ³²P-postlabeling in carp free-swimming in these tanks. ‘Clusters’ of lipophilic DNA adducts were detected, with five major and numerus minor adducts. Essentially a similar adduct pattern was found in the liver DNA of carp exposed to crude oil-polluted water. Diesel-2 adduct induction was observed slowly with a steady increase to > 3000 amol/μg DNA at day 12. After this time fish were transferred to clean water. Adduct levels continued to increase through day 17 (≈ 10,000 amol/μg DNA) despite the cessation of exposure, but a 30% and 80% decline was evident at day 22 and day 27, respectively. All major adducts were distinct from the known benzo[a]pyrene diolepoxide-dG. These results indicate that diesel-2 oil can cause extensive DNA damage in carp in vivo and the damage accumulates proportionately with time of exposure.     

Ninety-Day Inhalation Study in Rats, Using Aged and Diluted Sidestream Smoke from a Reference Cigarette: DNA Adducts and Alveolar Macrophage Cytogenetics

To study the genotoxic effects of subchronic exposure to environmentaltobacco smoke, Sprague-Dawley rats were exposed to 0, 0.1, 1.0,and 10 mg total particulate matter (TPM)/m³ of aged and dilutedsidestream smoke (ADSS) from 1R4F reference cigarettes 6 hrper day, 5 days a week for 13 weeks. DNA from lung, heart, larynx,bladder, and liver was tested for adduct formation by the ³²P-postlabelingassay after 28 (except bladder) and 90 days of exposure and90 days after cessation of exposure. In addition, alveolar macrophagesfrom animals exposed for 28 or 90 days were examined for chromosomalaberrations. Exposure-related DNA adducts were not observedin any tissue in any of the animals exposed to 0.1 or 1.0 mgTPM/m³. However, increased levels of DNA adducts with diagonalradioactive zones were observed in lung, heart, and larynx DNAof animals exposed to the highest concentration of ADSS (10mg TPM/m³). Adduct analyses with varying amounts of DNA fromlungs of mid-and high-exposure animals clearly indicate thatthe dose-response for DNA adduct formation is nonlinear. Theadduct levels were highest after 90 days of exposure and weresignificantly reduced in all target tissues 90 days after cessationof exposure. Chromosomal aberrations in alveolar macrophageswere not elevated in any group after 28 or 90 days of exposure.These results indicate a no-observed-effect-level (NOEL) ofat least 1.0 mg/m³ for DNA adduct formation in lung, heart,and larynx, and a NOEL of at least 10 mg/m³ for the inductionof chromosome aberrations in alveolar macrophages, under theconditions of this study.     

Flow cytometry, morphometry and histopathology as biomarkers of benzo[a]pyrene exposure in brown bullheads (Ameiurus nebulosus).

Brown bullheads were given a single intraperitoneal dose of 0, 5, 25 or 125 mg kg-1 benzo[a]pyrene (BaP), a carcinogenic polycyclic aromatic hydrocarbon, and evaluated over 18 months. Flow cytometric analyses of hepatocyte DNA content indicated an increase in DNA synthesis in BaP-exposed fish prior to day 14 post-exposure. Thereafter, all flow cytometric variables returned to initial levels. Histopathological evaluation of livers from fish sampled at 18 months revealed significant differences among treatments in the amount of hepatic macrophage ceroid pigmentation and basophilic staining intensity. No neoplasms or changes in blood cell DNA content were detected. Significant morphometric variations existed among fish, but differences between sexes overshadowed differences attributable to dose. Flow cytometry yielded no evidence of long-term DNA alterations from a single exposure to BaP; however, the differences detected by DNA analysis shortly after the toxic event suggest that flow cytometric cell cycle analysis may be useful for documenting continuing exposures.     

Development of a 32P-postlabeling method for the detection of 1, N2-propanodeoxyguanosine adducts of 2-hexenal in vivo

2-Hexenal is an alpha,beta-unsaturated carbonyl compound which forms cyclic 1,N2-propano adducts in vitro. The adduct formation in vivo was not reported by others to date. Because this type of adduct is considered promutagenic (2-hexenal is actually mutagenic and genotoxic) and humans are permanently exposed to this compound via vegetarian food, 2-hexenal may play a role in carcinogenicity. To improve the cancer risk assessment, we developed a new 32P-postlabeling technique for this compound and optimized the different steps of the postlabeling procedure. The results of the postlabeling methods are shown. A labeling efficiency of 35%, a recovery of 10% for the synthesized standards, and a detection limit of three 2-hexenal adducts per 10(8) nucleotides was achieved. After gavage of 500 mg/kg of body weight to male Fischer 344 rats, the respective DNA adducts were detected in rat liver DNA. With this study, we demonstrate in vivo adduct formation of 2-hexenal for the first time. Highest adduct levels were found 2 days after gavage, and after 4 days, the level was even higher than after 1 day. No adducts were detected 8 h after gavage. The respective adducts could not be found as a background in tissues of untreated rats or in calf thymus DNA at the limit of detection.     

Distribution of DNA adducts in the respiratory tract of rats exposed to diesel exhaust

Diesel exhaust, inhaled chronically at high concentrations, was previously found to be a pulmonary carcinogen in rats. The exhaust-induced tumors were located exclusively in the peripheral lung, although all of the respiratory tract tissues were exposed to the exhaust. The purpose of this study was to determine whether there were differences in the level of DNA adducts among the regions of the respiratory tract that paralleled the site of tumors. Groups of male F344/N rats were exposed 7 hr/day, 5 day/week for 12 weeks to diesel engine exhaust at a soot concentration of 10 mg/m3 or were sham-exposed to air. The maxilloturbinates, ethmoturbinates, trachea, left mainstem bronchus (airway generation 1), axial airway (airway generations 2-12), and peripheral lung tissue were dissected from the respiratory tract. DNA was isolated from the dissected samples and analyzed for the presence of adducts using the 32P-postlabeling assay. Chromatographic maps of DNA adducts demonstrated unique patterns of DNA adducts for each of the regions. The highest level of total DNA adducts occurred in peripheral lung tissue (approximately 20 adducts per 10(9) bases). The level of DNA adducts detected in the nasal tissues was about one-fourth to one-fifth that detected in peripheral lung. There were less than three adducts per 10(9) bases in each of the regions of the major conducting airways (i.e., trachea, bronchi, axial airway). In control rats, levels of DNA adducts ranged from one adduct per 10(9) bases (mainstem bronchi, axial airway) to about nine adducts per 10(9) bases (parenchyma). The data from this study indicate that higher levels of total DNA adducts and exhaust-induced adducts (i.e., exposed minus control adducts) were present in tissues where exhaust-induced tumors were located. These data suggest that DNA adduct levels in discrete locations of the respiratory tract may be good measures of the "effective dose" of carcinogenic compounds.     

Formation, persistence, and identification of DNA adducts formed by the carcinogenic environmental pollutant o-anisidine in rats

2-Methoxyaniline (o-anisidine) is an industrial and environmental pollutant causing tumors of urinary bladder in rodents. Here, we investigated the formation and persistence of DNA adducts in the Wistar rat. Using the (32)P-postlabeling method, three o-anisidine-derived DNA adducts were found in several organs of rats treated with a total dose of 0.53 mg o-anisidine/kg body wt (0.15, 0.18, and 0.2 mg/kg body wt ip in the first, second, and third day, respectively), of which the urinary bladder had the highest levels. At four posttreatment times (1 day, 13 days, 10 weeks, and 36 weeks), DNA adducts in bladder, liver, kidney, and spleen of rats were analyzed to study their persistence. In all time points, the highest total adduct levels were found in urinary bladder (39 adducts per 10(7) nucleotides after 1 day and 15 adducts per 10(7) nucleotides after 36 weeks) where 39% adducts remained. In contrast to the urinary bladder, no persistence was detected in other organs. All three DNA adducts were identified as deoxyguanosine adducts. When deoxyguanosine was reacted with the oxidative metabolite of o-anisidine, N-(2-methoxyphenyl)hydroxylamine, three adducts could be separated by high-performance liquid chromatography (HPLC) and were identified by mass spectroscopy and/or nuclear magnetic resonance spectrometry. All adducts are products of the nitrenium/carbenium ions, the reactive species generated from N-(2-methoxyphenyl)hydroxylamine. The major adduct was identified to be N-(deoxyguanosin-8-yl)-2-methoxyaniline. Using cochromatography on HPLC, this adduct was found to be identical to the major adduct generated by activation of o-anisidine in vitro and in vivo.