A romatic DNA adduct levels in human peripheral blood lymphocytes and total white blood cells by 32P postlabelling: need for validation

Long lived lymphocytes tend to have higher 32P postlabelling measured levels of adducts than short lived granulocytes in environmental and life style associated i.e. smoking exposures. With the aim of investigating this issue for occupational exposure to PAH and contributing to further validation of some technical aspects of the 32P postlabelling assay, two Italian laboratories analysed PAH-DNA adducts from lymphocytes and total white blood cells WBC . Seventy seven blood samples from coke oven workers employed at a steel plant located in Taranto, Southern Italy, and 14 samples from control subjects were collected. At the University of Padua, DNA was purified from peripheral blood lymphocytes PBL . Two years later, at the University of Bari, white blood cells WBC were isolated from replicate blood samples stored at- 80 C and DNA purified by the same method. In both cases, the nuclease P1 modified postlabelling assay was used to determine aromatic DNA adduct levels. The mean adduct levels were 5.13 3.37 Padua and 2.48 1.27 Bari per 108 nucleotides. Both laboratories observed large inter individual variations of adduct levels ranging from 0.09 to 18.93 per 108 nucleotides. Both the correlation and the agreement of the two sets of data were assessed. Slight correlation r = 0.39; p 0.01 and a poor level of agreement were found, the intra class correlation coefficient being equal to 0.05. Better correlation coefficient r = 0.54, p 0.01 and intra class correlation coefficient r = 0.50 were observed comparing only the adduct levels determined on the diagonal zone DRZ . Our findings seem to confirm the same divergence reported in the literature on DNA adduct levels between lymphocytes and granulocytes.     

A romatic DNA adduct levels in human peripheral blood lymphocytes and total white blood cells by 32P postlabelling: need for validation

Long lived lymphocytes tend to have higher 32P postlabelling measured levels of adducts than short lived granulocytes in environmental and life style associated i.e. smoking exposures. With the aim of investigating this issue for occupational exposure to PAH and contributing to further validation of some technical aspects of the 32P postlabelling assay, two Italian laboratories analysed PAH-DNA adducts from lymphocytes and total white blood cells WBC. Seventy seven blood samples from coke oven workers employed at a steel plant located in Taranto, Southern Italy, and 14 samples from control subjects were collected. At the University of Padua, DNA was purified from peripheral blood lymphocytes PBL. Two years later, at the University of Bari, white blood cells WBC were isolated from replicate blood samples stored at- 80 C and DNA purified by the same method. In both cases, the nuclease P1 modified postlabelling assay was used to determine aromatic DNA adduct levels. The mean adduct levels were 5.13 3.37 Padua and 2.48 1.27 Bari per 108 nucleotides. Both laboratories observed large inter individual variations of adduct levels ranging from 0.09 to 18.93 per 108 nucleotides. Both the correlation and the agreement of the two sets of data were assessed. Slight correlation r = 0.39; p 0.01 and a poor level of agreement were found, the intra class correlation coefficient being equal to 0.05. Better correlation coefficient r = 0.54, p 0.01 and intra class correlation coefficient r = 0.50 were observed comparing only the adduct levels determined on the diagonal zone DRZ. Our findings seem to confirm the same divergence reported in the literature on DNA adduct levels between lymphocytes and granulocytes.     

A comparison of DNA adduct formation in white blood cells and internal organs of mice exposed to benzo[a]pyrene, dibenzo[c,g]carbazole, safrole and cigarette smoke condensate

Measurement of tissue/cell DNA adducts represents a suitable monitor of carcinogen exposure because the majority of chemical mutagens/carcinogens react with DNA, forming covalent adducts, a key event in the initiation of chemical carcinogenesis. Investigations of DNA-adduct formation in vivo in white blood cells (WBC) versus target tissues, i.e. internal organs for most carcinogens, is expected to yield useful information about the suitability of WBC for biomonitoring and risk assessment. For this purpose, female ICR mice were given 0.4 mmole/kg benzo[a]pyrene (BP), 0.045 mmole/kg dibenzo[c,g]carbazole (DBC) or 2.47 mmole/kg safrole by oral gavage or 4 daily doses (equivalent to 3 cigarettes) of cigarette-smoke condensate (CSC) by topical application. At 24 h after dosing, DNA adducts were detected by a nuclease P1-enhanced 32P-postlabeling assay [M.V. Reddy and K. Randerath, Carcinogenesis, 7 (1986) 1543] in WBC and internal tissues treated with individual carcinogens, while CSC treatment elicited aromatic adducts in most tissues but not in WBC. Adduct patterns of WBC DNA were qualitatively similar to those of internal organs, but adduct amounts varied. BP, a systemic carcinogen, bound nearly as much to WBC DNA as to target-tissue DNA samples; whereas the liver carcinogens, DBC and safrole, bound to WBC DNA considerably less (22- and 51-fold, respectively) compared with liver DNA. The number of adducts in 10(7) nucleotides of WBC, liver, lung, kidney and spleen DNA, respectively, were: 2, 5, 3, 2 and 3 with BP; 6, 131, 6, 14 and 4 with DBC; 5, 238, 3, 5 and 0.6 with safrole. For CSC, these values were 0, 1 and 0.02 in WBC, lung and spleen, respectively. Our results show that carcinogen binding to WBC DNA does not reflect binding to target-tissue DNA in a quantitative sense for the carcinogens studied except for BP, and that WBC are not suitable surrogates for monitoring CSC exposure by DNA-adduct measurement after topical application. The CSC data in mice was consistent with the previous findings in humans that smokers' tissues but not WBC show smoking-related bulky/aromatic DNA adducts, as measured by 32P-postlabeling.     

Age-related induction and disappearance of carcinogen-DNA-adducts in livers of rats exposed to low levels of 2-acetylaminofluorene

It was investigated whether in vivo aging of rat liver is associated with changes in the induction and rate of disappearance of DNA damage. For this purpose 6- and 36-month-old rats were intraperitoneally injected with a single, low dose (5 mg/kg body wt.) of the model liver carcinogen 2-acetylaminofluorene (AAF). Using the 32P-postlabeling assay we found that N-(deoxyguanosin-8-yl)-2-aminofluorene (dG-C8-AF) was the major DNA-adduct formed. The minor adduct N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-C8-AAF) could only be detected after doses of 20 mg/kg or more. Quantitation of adduct levels at various time points after treatment indicated a rapid induction of AF-adducts, which were already present at 6 h after treatment. The subsequent loss of AF-adducts was relatively slow, as was indicated by the presence of a substantial amount of AF-adducts as late as 21 days after treatment. Slight age-related differences in the pattern of induction and disappearance of AF-adducts and a somewhat higher level of persisting lesions in old than in young rats were observed.     

Lack of DNA single-strand breaks in rat liver cells exposed to 4-acetylaminofluorene, in vivo and in vitro

The induction of primary DNA damage by the non-carcinogen 4-AAF was reinvestigated in liver cells by comparison with the carcinogen 2-AAF. DNA alkaline elution showed the appearance of single-strand breaks in total liver DNA of rats 4 h after gavage with 200 mg/kg of 4-AAF. The decrease in hepatocyte viability and yield observed in these livers after collagenase perfusion indicated a cytotoxic effect of 4-AAF treatment. Viable hepatocytes isolated from 4-AAF-treated rats as well as hepatocytes from normal rats treated with 4-AAF in vitro did not present DNA single-strand breaks.     

The organ-specific induction of DNA adducts in 2-acetylaminofluorene-treated rats, studied by means of a sensitive immunochemical method

Exposure of cells to chemical carcinogens and mutagens may result in the formation of DNA adducts, which can give rise to mutations in the genome and to cellular transformation. Methods to measure DNA-adduct formation may be useful for ‘biomonitoring’, to establish exposure of laboratory animals or humans to DNA-damaging agents. For such purposes, immunochemical methods appear to be suitable, because they allow sensitive detection and quantification of DNA adducts in small amounts of sample in a non-radiolabelled form. We have worked out optimal conditions for the detection of DNA adducts by means of competitive enzyme-linked immunosorbent assay (ELISA). This technique involves interaction of soluble antigen, immobilized antigen and antibody. It appeared that the sensitivity of the competitive assay can be improved by lowering the amount of immobilized antigen, adsorbed to the wall of the plastic reaction vessel. On the basis of these observations, suitable conditions were selected for a sensitive quantitative assay of adducts in DNA isolated from various organs of rats, treated (p.o.) with the liver carcinogen 2-acetylaminofluorene (2-AAF). Under the conditions of these experiments, the available rabbit antiserum recognizes the guanosine-AAF adduct with high specificity. A time- and dose-dependent induction of AAF adducts could be measured in liver DNA from exposed rats, whereas the amount of adducts in DNA from spleen and nucleated blood cells remained below the detection limit (1 adduct/10⁸ nucleotides). The implications of these findings with respect to the relevance of blood cell biomonitoring for target cell exposure are discussed.     

Acceleration of the onset of liver regeneration by carnitine in partially hepatectomized rats

Immediately and 6 h after removal of 70% of the liver tissue, rats were treated with L-carnitine (Carnitene, Sigma-Tau, Italy) and received an injection of 100, 200 or 1,000 mg/kg b.w. into their femoral vein. The control rats were given the same volume of saline solution. The rats were sacrificed 18, 21, 24 or 30 h after the operation. The development of liver regeneration was evaluated from the incorporation of 14C-thymidine into DNA and from the hepatocyte mitiotic activity. In rats given carnitine in a dose of 100 or 200 mg/kg b.w. significantly higher DNA specific activity values were found 18 and 21 h after partial hepatectomoy and higher hepatocyte mitotic activity values after 30 h. In rats given carnitine in a dose of 1,000 mg/kg b.w., DNA specific activity values 21 h after partial hepatectomy were lower than in the control group. We conclude that L-carnitine, in a dose of 100 or 200 mg/kg b.w. has an enhancing effect on the onset of liver regeneration after 70% hepatectomy.     

Chemopreventive effect of bilberry (Vaccinium myrtillus) against cisplatin-induced oxidative stress and DNA damage as shown by the comet assay in peripheral blood of rats

The aim of this study was to evaluate the chemopreventive effect of bilberry on cisplatin induced oxidative stress and DNA damage in rat blood. Twenty-one female Wistar-Albino rats were divided into three groups: group I — untreated; group II — treated with cisplatin (single dose 7.5 mg/kg b.w.); and group III — treated with cisplatin (single dose 7.5 mg/kg b.w.) and bilberry (200 mg/kg b.w. for 10 days). Antioxidant enzyme systems including superoxide dismutase, catalase, glutathione peroxidase and the level of malondialdehyde (MDA) that might occur on erythrocytes have been determined and single cell gel electrophoresis (comet) was utilized to evaluate the DNA damage in lymphocytes. Treatment with cisplatin has increased the levels of MDA and decreased antioxidant enzymes in erythrocytes. Comet assay showed significantly higher values at dose of 7.5 mg/kg cisplatin as the result of oxidative DNA damage when compared to the control group. Cisplatin treatment with bilberry resulted in a highly significant (P < 0.05) decreased in the lymphocytes DNA when compared to the cisplatin group. Bilberry has been effective on antioxidant enzyme systems and MDA level and significantly decreased the comets. Our results indicate that bilberry is capable of preventing genotoxic and cytotoxic damage caused by cisplatin in peripheral blood cells in rats.     

Formation of adducts by bisphenol A, an endocrine disruptor, in DNA in vitro and in liver and mammary tissue of mice

Endocrine disruptors (EDs) represent a major toxicological and public health issue, and the xenoestrogen bisphenol A (BPA) has received much attention due to its high production volume and widespread human exposure. Also, due to its similarity to diethylstilbestrol, a known human carcinogen, BPA has been investigated for its genotoxic and carcinogenic properties, but the results have been either inconclusive or controversial. Metabolically activated BPA has previously been shown to form DNA adducts both in vitro and in rat liver. The present study was designed (a) to assess the sensitivity threshold of DNA-adduct detection by ³²P-postlabelling in an acellular system and (b) to evaluate the formation of DNA adducts in both liver and mammary cells of female CD-1 mice receiving BPA in their drinking water (200 mg/kg body weight) for eight consecutive days. The reaction of BPA with calf thymus DNA, in the presence of S9 mix, resulted in a dose-dependent formation of multiple DNA adducts, with a detection limit of 10 ng of this ED under our experimental conditions. Administration of BPA to mice confirmed that DNA adducts are formed in liver (3.4-fold higher levels than in controls). In addition, new evidence is provided that DNA adducts are formed in target mammary cells (4.7-fold higher than in controls). Although DNA adducts do not necessarily evolve into tumours or other chronic degenerative diseases, the formation of these molecular lesions in target mammary cells may bear relevance for the potential involvement of BPA in breast carcinogenesis.     

A review of the dose-response induction of DNA adducts by aflatoxin B1 and its implications to quantitative cancer-risk assessment

The induction of DNA adducts by aflatoxin B₁ in the liver has been extensively reviewed in a quantitative cancer-risk assessment of aflatoxins (CDHS, 1990). Rat is the most sensitive species for aflatoxin tumorigenesis and liver is the most sensitive site. In vitro DNA-adduct studies were mostly on adduct identification and specificity of binding. In vivo studies provided dose-response relationship of aflatoxin B₁, binding to DNA and DNA-adduct formation. Most in vivo studies were conducted in rats. The dose-response curves of DNA-adduct induction after ingestion or injection treatments in this species were reviewed. A linear dose-response relationship was observed in both injection and ingestion studies at low doses. For cancer-risk assessment, this observation is consistent with the assumption of the linear dose-response risk-assessment model for genotoxic agents, and justifies the use of this model for quantitative cancer-risk assessment for aflatoxins.     

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.     

Biomarkers of marine pollution observed in species of mullet living in two eastern Mediterranean harbours

The activities of enzymes associated with xenobiotic metabolism and or oxidative processes, and the levels of aromatic DNA adducts, have been determined in the livers of grey mullet Oedalechilus labeo and Lisa ramada living in two eastern Mediterranean harbours. Glutathione peroxidase GSH P activity was 2.5 times higher 9 IU g-1 liver and glutathione reductase GSSG R activity was twice as high 2.5 IU g-1 liver in fish from the more polluted harbour at Mersin than in the harbour near Erdemli. Superoxide dismutase SOD activity was 25 lower 4.3 IU g-1 liver in the more polluted harbour. The concentrations of glutathione and malondialdehyde varied both with species and environment by a factor of 2.5-3. DNA adducts in liver were determined by 32P postlabelling. In Oedalechilus labeo in the more polluted harbour, adduct levels were 258 21 adducts per 108 nucleotides mean SE; two groups of Lisa ramada were distinguished having 261 48 and 30 6 adducts per 108 nucleotides, respectively. The average adduct level in a group of mullet of mixed species in the less polluted harbour was 3.3 2.3 adducts per 108 nucleotides. The results illuminate the ability of mullet to live in contaminated marine environments, and show that enzyme activities and liver DNA adduct levels can serve as indicators of marine pollution.     

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.     

DNA damage and DNA adduct formation in rat tissues following oral administration of acrylamide

Acrylamide is present as a contaminant in the human diet in heated food products. It has been found to be carcinogenic in laboratory rats and has been classified as probably carcinogenic in humans. In order to clarify the possible involvement of a primary genotoxic mechanism in acrylamide-induced carcinogenicity, both the presence of DNA damage, measured by the comet assay, and the formation of N7-(2-carbamoyl-2-hydroxyethyl)guanine (N7-GA-Gua) and N3-(2-carbamoyl-2-hydroxyethyl)adenine (N3-GA-Ade), derived from reaction of the active metabolite glycidamide (GA) with the DNA, analyzed by LC/MS/MS, were assessed in selected rat tissues. Rats were administered with single oral doses of acrylamide (18, 36 or 54 mg/kg body weight (b.w.) and the organs (blood leukocytes, brain, bone marrow, liver, testes and adrenals) were sampled at different times after treatment. Results from GA-induced DNA adduct measurements indicated a relatively even organ distribution of the adducts in brain, testes and liver. Organ-specificity in acrylamide carcinogenesis can therefore not be explained by a selective accumulation of GA-DNA adducts in the target organs, at least not after a single dose exposure. The DNA adduct profiles and half-lives were similar in the different organs; except that the N3-GA-Ade adduct was more rapidly removed from tissues than the N7-GA-Gua adduct. Increased extent of DNA migration, as measured by the in vivo rat comet assay, was found in brain and testes, and these specific results seem to be in accordance with the known organ-specificity in acrylamide carcinogenesis in rat. Only weak and transient DNA damage was recorded in the liver, bone marrow and adrenals. The DNA-damaging effect of the compound observed in the blood leukocytes could be a simple biomarker of acrylamide exposure and genotoxicity.     

DNA adducts in relation to lung tumour outcome are not markers of susceptibility following a single dose treatment of SWR, BALB/c and C57BL/6J mice with N-nitrosodiethylamine

The formation of DNA adducts by the covalent binding of genotoxic chemicals to DNA represents a valuable marker for assessing exposure to carcinogens but as yet the role of DNA adducts as a biomarker of carcinogenic susceptibility still needs to be clearly ascertained. To address this question an animal study was instigated using mice (SWR (high), BALB/c (intermediate) and C57BL/6J (low)) varying in their susceptibility to lung carcinogenesis. Groups of animals from each strain were dosed with a single intraperitoneal injection of saline or N -nitrosodiethylamine (NDEA) at 15 or 90 mg kg^-1 body weight. Lung and liver tissues were removed at different time points following dosing. Further groups of mice dosed with the same regime had urine samples collected 24 h post dosing and were then left up to 18 months to allow for the development of tumours. Immunoslot-blot analysis was used for the determination of N-7 ethylguanine (N-7EtG) and O⁶ ethylguanine (O⁶EtG) adduct levels in the DNA from the tissues and gas chromatography-mass spectrometry (GC-MS) was used to determine N-3 ethyladenine (N-3EtA) adduct levels in the urine samples. Levels of alkyltransferase (ATase) were also determined in the tissues. The results showed that the DNA adduct levels and persistence were similar across the three strains of mice following dosing with 15 and 90 mg kg^-1 NDEA. High levels of adducts were observed in the urine of the BALB/c strain, implying an increased metabolic or repair capacity in this strain. However there were no differences in the levels of ATase in the lung and liver of the three strains of mice following dosing with 15 mg kg^-1 NDEA. The incidence of tumours in C57BL/6J mice was lower compared with the other two strains and showed a dose dependent increase. The results from this study show that the differences in susceptibility to lung carcinogenesis between the three strains of mice do not appear to be linked to the formation of the two adducts detected. These results imply that dosing with NDEA resulted in toxicity which may have led to cell death and induction of tumours by compensatory cell proliferation. Although these results do not allow decisive conclusions to be drawn concerning the relationship between total levels of DNA adducts and differences in carcinogenic susceptibility for the three strains of mice it is clear that the increased presence of a DNA adduct in the target tissue increases the likelihood of tumour development.     

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)     

DNA adducts in relation to lung tumour outcome are not markers of susceptibility following a single dose treatment of SWR,BALB/c and C57BL/6J mice with N-nitrosodiethylamine

The formation of DNA adducts by the covalent binding of genotoxic chemicals to DNA represents a valuable marker for assessing exposure to carcinogens but as yet the role of DNA adducts as a biomarker of carcinogenic susceptibility still needs to be clearly ascertained. To address this question an animal study was instigated using mice (SWR (high), BALB/c (intermediate) and C57BL/6J (low)) varying in their susceptibility to lung carcinogenesis. Groups of animals from each strain were dosed with a single intraperitoneal injection of saline or N -nitrosodiethylamine (NDEA) at 15 or 90 mg kg^-1 body weight. Lung and liver tissues were removed at different time points following dosing. Further groups of mice dosed with the same regime had urine samples collected 24 h post dosing and were then left up to 18 months to allow for the development of tumours. Immunoslot-blot analysis was used for the determination of N-7 ethylguanine (N-7EtG) and O⁶ ethylguanine (O⁶EtG) adduct levels in the DNA from the tissues and gas chromatography-mass spectrometry (GC-MS) was used to determine N-3 ethyladenine (N-3EtA) adduct levels in the urine samples. Levels of alkyltransferase (ATase) were also determined in the tissues. The results showed that the DNA adduct levels and persistence were similar across the three strains of mice following dosing with 15 and 90 mg kg^-1 NDEA. High levels of adducts were observed in the urine of the BALB/c strain, implying an increased metabolic or repair capacity in this strain. However there were no differences in the levels of ATase in the lung and liver of the three strains of mice following dosing with 15 mg kg^-1 NDEA. The incidence of tumours in C57BL/6J mice was lower compared with the other two strains and showed a dose dependent increase. The results from this study show that the differences in susceptibility to lung carcinogenesis between the three strains of mice do not appear to be linked to the formation of the two adducts detected. These results imply that dosing with NDEA resulted in toxicity which may have led to cell death and induction of tumours by compensatory cell proliferation. Although these results do not allow decisive conclusions to be drawn concerning the relationship between total levels of DNA adducts and differences in carcinogenic susceptibility for the three strains of mice it is clear that the increased presence of a DNA adduct in the target tissue increases the likelihood of tumour development.     

32P-analysis of DNA adducts in somatic and reproductive tissues of rats treated with the anticancer antibiotic, mitomycin C

Mitomycin C (MMC) is a clinically used drug with mutagenic and antitumor activities, presumably elicited through its covalent binding to DNA, however, little is known about MMC binding to DNA in vivo. A 32P-postlabeling method that does not require radiolabeled test compounds was employed here to study the formation of DNA adducts in somatic and reproductive tissues of rats 24 h after an i.p. dose of 9 mg/kg MMC. Among 14 tissues studied in female rats, MMC-DNA adduct levels were within a 2-fold range in 11 tissues, i.e. bladder, colon, esophagus, heart, kidney, liver, lung, ovary, pancreas, small intestine and stomach (minimum levels of 9.6-21.9 adducts per 10(7) N). Three other tissues, i.e. brain, spleen and thymus, exhibited lower adduct levels (0.2 5.4 and 1.4 adducts, respectively, per 10(7) N). Liver DNA adduct levels were 32% lower in male than in female rats. Testicular DNA contained 2.5 adducts per 10(7) N, i.e. 5.3 times less than ovarian DNA. 32P-labeled adduct patterns were qualitatively similar among the different tissues and consisted of 10 adducts, one of which comprised 71 (+/- 5)% of the total. All these adducts were chromatographically identical to adducts formed by the reaction of chemically reduced MMC with DNA in vitro, demonstrating that metabolic activation of MMC occurred via reduction. Using homopolydeoxyribonucleotides modified with MMC, in vivo adducts were shown to be mostly (greater than 90%) guanine derivatives and small amounts of adenine, cytosine and thymine products. Most of the adducts appeared to be monofunctional derivatives of DNA nucleotides. Dose-dependent MMC-DNA adduct formation was determined in rat liver over an 82-fold range of MMC administered (0.11-9.0 mg/kg). The lowest dose level studied was 4.5 times lower than the recommended single dose for human cancer chemotherapy (20 mg/m2). Thus, these results predict that 32P-postlabeling methodology is suitable to monitor and quantify DNA adducts in tissue biopsies of patients receiving MMC chemotherapy.     

Formation and persistence of benzo[a]pyrene-diolepoxide-DNA adducts in liver of English sole (Parophrys vetulus)

The formation of DNA adducts from the carcinogenic environmental pollutant benzo[a]pyrene (BaP) was investigated in liver of English sole (Parophrys vetulus), a fish species that exhibits a high prevalence of liver neoplasms in several polycyclic aromatic hydrocarbon (PAH)-contaminated areas of Puget Sound, WA. Analysis by the 32P-postlabeling assay of hepatic DNA digests from English sole exposed parenterally to BaP showed the presence of BaP-diol epoxide (BaPDE)-DNA adducts. When English sole were injected with 2-15 mg BaP/kg body wt., one major adduct was detected and was identified as the anti-BaPDE-DNA adduct. Moreover, in English sole sampled at 1, 28 and 60 days post-exposure to 15 mg BaP/kg body wt., there was no significant change in the level of the anti-BaPDE-DNA adduct. The autoradiographs of 32P-labeled hepatic DNA digests from fish exposed to 100 mg BaP/kg body wt. showed an elongated spot suggesting the presence of more than one adduct. Chromatography on large polyethyleneimine sheets (20 x 20 cm) showed 2 spots with the same chromatographic characteristics as those of syn- and anti-BaPDE-deoxyguanosine adduct standards. Mild acid hydrolysis of hepatic DNA of English sole, exposed to 100 mg BaP/kg body wt., also revealed the presence of tetrols derived from both anti- and syn-BaPDE, thus confirming the presence of syn- and anti-BaPDE. In fish exposed to 2-100 mg BaP/kg body wt., a linear (0.996) dose response for anti-BaPDE-DNA adduct formation was observed. The results from this study offer the first direct evidence for the formation of the suspected ultimate carcinogen, BaPDE, in liver of English sole exposed to BaP in vivo and thus further support the hypothesis that exposure to PAHs is an important factor in the etiology of hepatic neoplasms in English sole from contaminated sites.