Formation of DNA adducts from N-acetoxy-2-acetylaminofluorene and N-hydroxy-2-acetylaminofluorene in rat hemopoietic tissues in vivo

Administration of [ring-3H]-N-acetoxy-2-acetylaminofluorene (10 mg/kg i.v.) to male F344 rats resulted in substantial binding of [ring-3H]-N-acetoxy-2-acetylaminofluorene to DNA isolated from bone marrow [20.3 +/- 1.7 (SD) pmol/mg DNA] and spleen (23.6 +/- 5.8 pmol/mg DNA) compared to liver (39.4 +/- 2.1 pmol/mg DNA) and kidney (27.1 +/- 1.0 pmol/mg DNA) 2 h after dosing. High-performance liquid chromatography analyses of trifluoroacetic acid hydrolyzed DNA from bone marrow and spleen revealed the presence of N-(guanin-8-yl)-2-aminofluorene as the major adduct comprising more than 80% of total adducts, while N-(guanin-8-yl)-2-acetylaminofluorene and ring opened derivatives of N-(guanin-8-yl)-2-aminofluorene were only minor adducts. Dose dependent binding of [ring-3H]-N-hydroxy-2-acetylaminofluorene (N-OH-AAF) to DNA and formation of individual adducts in spleen and bone marrow was observed at a dose range of 1.0-10.0 mg/kg. There was a 3- and 6-fold more DNA adduct formation in bone marrow and spleen, respectively, following treatment with [ring-3H]-N-acetoxy-2-acetylaminofluorene compared to N-OH-AAF. However, the pattern of DNA adducts formed was similar. Pretreatment of rats with the cytotoxic agent 5-fluorouracil (150 mg/kg i.p.), which causes transient depletion of hemopoietic cells, on days -10, -7, -4, -2, and -1 prior to the administration of [ring-3H]-N-OH-AAF (10 mg/kg) on day 0 resulted in different levels of N-OH-AAF binding to spleen and bone marrow DNA without altering the pattern of DNA adducts compared to that in control animals. These data suggest a possible existence of a target cell population for N-OH-AAF and perhaps other aromatic amines and amides in both bone marrow and spleen of F344 rat.     

The role of specific DNA adducts in the induction of micronuclei by N-hydroxy-2-acetylaminofluorene in rat liver in vivo

N-Hydroxy-Z-acetylaminofluorene (N-OH-AAF) was administeredi.p. to male Wistar rats 17 h after partial hepatectomy. Hepatocyteswere analyzed for the presence of micronuclei 7 h, 1, 2, 3 and4 days after injection. N-OH-AAF treatment resulted in a highfrequency of micronucleated hepatocytes at days 3 and 4 (19.5and 19.6 respectively). The frequency of micronucleated hepatocyteswas not increased above control values when hepatocytes wereisolated as early as 7 h, 1 or 2 days after injection. Pretreatmentwith the sulfotransferase inhibitor pentachlorophenol (PCP)45 min before injection of N-OH-AAF almost completely preventedthe formation of micronuclei by N-OH-AAF. Parallel biochemicalstudies indicated that inhibition of sulfation of N-OH-AAF byPCP pretreatment prevented the formation of the N-acetylatedDNA adducts iV-deoxyguanosin-8-yl-AAF and 3-deoxyguanosin-N²-yl-AAFby {small tilde}85%. Total adduct formation to DNA was, however,not lowered because of an increase in the formation of the deacetylatedadduct, N-deoxy-guanosin-8-yl-AAF. The lower frequency of micronucleatedhepatocytes observed in the group pretreated with PCP, did notresult from less proliferative activity in this group as comparedto the group treated with N-OH-AAF alone. Therefore, the decreasein the formation of micronuclei indicates that PCP preventsthe clastogenic damage caused by N-OH-AAF. It is concluded thatthe clastogenicity of N-OH-AAF in rat liver is related to theformation of N-acetylated DNA adducts (i.e. N-deoxyguanosin-8-yl-AAFand/or 3-deoxy-guanosin-N²-yl-AAF) and is not related to theformation of the deacetylated DNA adduct N-deoxyguanosin-8-yl-AF.     

Quantification of adducts formed in DNA treated with N-acetoxy-2-acetylaminofluorene or N-hydroxy-2-aminofluorene: comparison of trifluoroacetic acid and enzymatic degradation

We have examined two methods of preparation of DNA adducts fromX174 RF DNA modified by [³H]N-acetoxy-2-acetylaminofluorene([³H]NA-AAF) or N-hydroxy-2-amino- fluorene ([³]N-OH-AF). Hydrolysisby enzymes (DNase I, snake venom phosphodiesterase and alkalineor acid phosphatase) and subsequent reverse phase h.p.l.c. ofX174 RF DNA treated with [³H]NA-AAF yielded 73% N-(deoxyguanosin-8-yl)-2-acetylaminofluorene(dG-C8-AAF), 7% 3-(deoxyguanosin-N²-yl)-2acetylaminofluorene(dG-N²-AAF), and a peak of unidentified radioactivity (13%).When [³H]N-OH-AF modified X174 DNA was analyzed, both N-(deoxyguanosin-8-yl)-2-aminofluorene(dG-C8-AF) and a large percentage of the imidazole ring-openedderivative and unidentified products were found. In contrast,when anhydrous trifluoroacetic acid (TFA) was used to degradethese DNAs, we found for the [³H]NA-AAF modified DNA 86% N-(guanin-8-yl)-2-acetylaminofluorene(G-C8-AAF) and 6% 3-(guanin-N²-yl)-2acetylaminofluorene (G-N²-AAF),while for [³H]N-OH-AF modified DNA only the N-(guanin-8-yl)-2-aminofluorene(G-C8-AF) was found. When DNA was prepared from human fibroblaststreated with [³H]NA-AAF, only the G-C8-AF product was obtained.Thus, anhydrous TFA solvolysis followed by reverse phase h.p.l.c.is a rapid and convenient method to obtain quantitative yieldsof DNA adducts formed with acetylamino-fluorene and relatedcompounds: quantification by this method prevents loss of G-N²adducts, the conversion of AAF adducts to AF adducts, and theproduction of ring opened products in guanine residue.     

Formation and persistence of DNA adducts from the carcinogen N-hydroxy-2-acetylaminofluorene in rat mammary gland in vivo

The rat mammary carcinogen, N-hydroxy-2-acetylamino-fluorene(N-hydroxy-2-AAF), has been proposed to be metabolically activatedby mammary cytosolic N,O-acetyltransferase to a DNA bindingspecies. To test this hypothesis, adult female Sprague-Dawleyderived CD rats were treated, l.p., with 4.0 mg/kg [ring-³H]N-hydroxy-2-AAF.After 4 h, 1, 3, 14, and 28 days, the animals were killed, themammary epithelium DNA was isolated and the carcinogen-deoxyribonucleosideadducts present were analyzed by high pressure liquid chromatography.At each time, only one adduct was detected and it was chromatographicallyidentical to N-(deoxyguanosin-8-yl)-2-aminofluorene. The levelof the adduct was maximal at 4 h (1.5 adducts/10⁶ nucleotides)and then decreased, following first order kinetics with a t_1/2of 14.2 days. The detection of a single non-acetylated aminofluoreneadduct is consistent with N,O-acyltransferase being involvedin the metabolic activation of N-hydroxy-2-AAF in the rat mammarygland.     

The binding of N-hydroxy-2-acetylaminofluorene to DNA and repair of the adducts in primary rat hepatocyte cultures

Primary cultures of rat hepatocytes were exposed to [ring-³H]-N-hydroxy-2-acetylaminofluorene(N-OH-AAF) for 4 h, and the RNA and DNA nucleoside adducts wereisolated and identified by h.p.l.c. The DNA adducts were shownto be N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-C8-AAF),N-(deoxyguanosin-8-yl)-2-aminofluorene (dG-C8-AF), and 3-(deoxy-guanosin-8-yl)-2-acetylaminofluorene(dG-N2-AAF), while the RNA adducts were N-(guanosin-8-yl)-2-acetyl-aminofluorene,and N-(guanosin-8-yl)-2-aminofluorene. The removal of theseadducts was measured up to 38 h following the cessation of exposureof the hepatocytes to N-OH-AAF. The dG-C8-AAF adduct was removedwith a half-life of approximately 10 h, while dG-N²-AAF anddG-C8-AF remained constant for 14 h, followed by a slow rateof removal. The dG-C8-AAF adduct initially composed about 60%of the total DNA adducts of primary hepatocytes in contrastto the 20% found in liver in vivo. The formation of the 3 DNAadducts and the different rates of repair indicate that primarycultured rat hepatocytes may be a valuable system to study initiationof liver carcinogenesis by N-OH-AAF.     

Induction of DNA strand breaks in chronic lymphocytic leukemia following treatment with 2'-deoxycoformycin in vivo and in vitro

Four patients with refractory chronic lymphocytic leukemia were treated with the adenosine deaminase inhibitor, 2'-deoxycoformycin, and initially received 4 mg/m2 i.v. weekly. Clinical responses to therapy varied: Patient A had a minimal response; whereas Patient D showed an 85% decrease in lymphocyte count at 2 wk; and Patients B and C had intermediate responses. The pretreatment mononuclear cell adenosine deaminase activities, which ranged from 1.6 to 44.6 nmol adenosine/h/10(6) cells, decreased to approximately 1 nmol adenosine /h/10(6) cells 24 h following 2'-deoxycoformycin, and increased to 15 to 50% of the pretreatment activity prior to the second drug treatment. The clinical response to 2'-deoxycoformycin was unrelated to the pre- or posttreatment adenosine deaminase activities or to the rate of return of enzyme activities following treatment. The plasma deoxyadenosine levels and the leukemic cell dATP concentrations rose slightly with therapy, but there was no correlation between the magnitude of increase and clinical response. No significant levels of DNA strand breaks were observed in the leukemic cells following treatment, although the NAD levels decreased slightly in two patients. When peripheral mononuclear cells from the patients and two controls were incubated in vitro for 24 h with 2'-deoxycoformycin and increasing concentrations of deoxyadenosine, a concentration-dependent increase in dATP and decrease in NAD were observed in both the patients and normals. The normal cells, and cells from two patients, developed a significant number of DNA strand breaks. However, there was no relationship between the formation of DNA breaks and the degree of accumulation of dATP or depletion of NAD, or between any of these changes and subsequent clinical responses to 2'-deoxycoformycin. Based on this study, it appears that the antitumor activity of 2'-deoxycoformycin in chronic lymphocytic leukemia is unrelated to the induction of DNA strand breaks or to changes in the levels of dATP or NAD in the leukemic cells.     

Persistence of DNA adducts in rat liver and kidney after multiple doses of the carcinogen N-hydroxy-2-acetylaminofluorene

Male and female Sprague-Dawley rats were treated by gastric intubation either 1, 2, 3, or 4 times at biweekly intervals with 10-mg/kg doses of the hepatocarcinogen of N-[ring-3H]-hydroxy-2-acetylaminofluorene. Then either 1 or 14 days following the last treatment, the concentrations of 2-aminofluorene and 2-acetylaminofluorene adducts in liver and kidney DNA were established. 2-Acetylaminofluorene adducts were found in male rat liver DNA only. The C-8-acetylated adduct, N-(deoxyguanosin-8-yl)-2-acetylaminofluorene, was detected only on the day following treatment at a concentration between 1.0 and 2.4 pmol/mg DNA. A second acetylated adduct, 3-(deoxyguanosin-N2-yl)-2-acetylaminofluorene, was found at both 1 and 14 days after treatment and, as a result, increased in concentration with repeated doses, from 0.2 pmol/mg DNA after one dose to 2.8 pmol/mg DNA after four treatments. The major adduct detected in male rat liver and the only adduct found in female rat liver and in kidney from either sex was N-(deoxyguanosin-8-yl)-2-aminofluorene. This adduct was slowly lost from the DNA and therefore increased in concentration with repetitive treatments as follows: male liver, 4.0 to 9.4 pmol/mg DNA; female liver, 11.4 to 30.6 pmol/mg DNA; male kidney, 1.1 to 1.8 pmol/mg DNA; and female kidney, 1.8 to 17.7 pmol/mg DNA. These data indicate that certain DNA adducts can accumulate in both target and non-target tissues and therefore suggest that persistence of DNA adducts per se is not sufficient for tumor induction.     

Ascorbate potentiates DNA damage by 1-methyl-1-nitrosourea in vivo and generates DNA strand breaks in vitro

Ascorbic acid (vitamin C) is an important intracellular reducingagent. It also has been suggested to be (i) a protective agentagainst development of cancer, (ii) a therapeutic agent formalignancies and (iii) a mutagen. We have found that high concentrationsof ascorbate leads to DNA damage in several in vivo and in vitrosituations. Guinea-pigs receiving oral 1-methyl-1-nitrosourea(MNU) were used as a whole animal model. Administration of sodiumascorbate prior to MNU increased strand breakage in pancreaticDNA. Concentrations of ascorbate >0.5 mM increased the frequencyof DNA strand breaks caused by MNU in both L1210 murine leukemiacells and guinea-pig pancreatic cells in tissue culture; ascorbatealone led to DNA strand breaks in the latter cells. Investigationsof the mechanism of DNA damage were carried out with purifiedDNA. Ascorbate produced single- and double-strand breaks inplasmid DNA. Cleavage was catalyzed by copper (II), inhibitedby catalase and blocked by the presence of thiols. We concludethat superoxide and hydrogen peroxide produced during the oxidationof ascorbate leads to generation of hydroxyl free radicals thatcan mediate DNA strand scissions and potentiate the effectsof alkylating carcinogens.     

Role of sulfation in the formation of DNA adducts from N-hydroxy-2-acetylaminofluorene in rat liver in vivo. Inhibition of N-acetylated aminofluorene adduct formation by penta-chlorophenol

N-Hydroxy-2-acetylaminofluorene (N-OH-AAF) is metabolicallyconverted into reactive N, O-esters which are capable of formingcovalent adducts with DNA in rat liver in vivo. The effect ofinhibiting one of the proposed pathways, N-O-sulfation, on DNAadduct formation was studied by using a specific sulfotransferaseinhibitor, pentachlorophenol. Rats were pretreated with pentachlorophenoland, after 45 min, N-OH-AAF was administered. Four hours afterdosing the animals were sacrificed and hepatic DNA was isolated.In DNA from control livers two acetylaminofluorene-and one amino-fluorene-substituteddeoxyguanosine adducts were found. The acetylaminofluorene derivatives,N-(deoxy-guanosin-8-yl)-2-acetylaminofluorene and 3-(deoxy-guanosin-N²-yl)-2-acetylaminofluorene,accounted for 40% of the total binding in the hydrolyzed DNA.The aminofluorene adduct, N-(deoxyguanosin-8-yl)-2-aminofluorene,accounted for the remainder. In rats that were pretreated withpentachlorophenol, total DNA binding was decreased by 26%. Thesame three adducts were found, but the acetylaminofluorene adductswere now only 13% of the total, while the aminofluorene adductaccounted for 87%. The absolute amount of aminofluorene adductwas not altered as compared to control rats. These data demonstratethe involvement of N-O-sulfation in carcinogen - DNA bindingand indicate that at least 70% of the acetylaminofluorene boundto deoxyguanosine in rat liver DNA, in vivo, is formed throughN-O-sulfation of N-OH-AAF.     

Sulfation-dependent formation of N-acetylated and deacetylated DNA adducts of N-hydroxy-4-acetylaminobiphenyl in male rat liver in vivo and in isolated hepatocytes.

Administration of 3H-labeled N-hydroxy-4-acetylaminobiphenyl (N-OH-AABP) to male Wistar rats with or without prior partial hepatectomy (PH) resulted in covalent binding of 3H activity to liver macromolecules. Pretreatment with the sulfotransferase inhibitor pentachlorophenol (PCP) 45 min before administration of the arylhydroxamic acid strongly decreased the covalent binding. Analysis of aminobiphenyl adducts after TFA hydrolysis of DNA and RNA showed that PCP decreased the formation of both the N-acetylated adduct N-[deoxy)guanosin-8-yl)-4-acetylaminobiphenyl [(d)G-C8-AABP] and the deacetylated adduct N-[deoxy)-guanosin-8-yl)-4-aminobiphenyl [(d)G-C8-ABP] by 60-80%. In incubations with hepatocytes from male Wistar or Sprague-Dawley rats, omission of inorganic sulfate also strongly decreased the covalent binding of 3H-labeled N-OH-AABP to RNA and protein. Analysis of RNA adducts showed a 70-80% decrease in the formation of G-C8-ABP in the absence of sulfate. Another, as yet unidentified, adduct was only slightly decreased. Similar results were obtained with the structurally related carcinogen N-hydroxy-4'-fluoro-4-acetylaminobiphenyl (N-OH-FAABP). Pretreatment with PCP decreased the incidence of gamma-glutamyltranspeptidase-positive foci in the liver of male rats when analyzed 30 days after a single injection of N-OH-AABP or N-OH-FAABP by 60 and 80% respectively. Thus, both N-acetylated and deacetylated RNA and DNA adducts of N-OH-AABP in rat liver are formed by sulfation and this metabolic activation pathway is responsible for the formation of genotoxic metabolites involved in the generation of preneoplastic cells.     

Correlation between clastogenicity and promotion activity in liver carcinogenesis by N-hydroxy-2-acetylaminofluorene, N-hydroxy-4'-fluoro-4-acetylaminobiphenyl and N-hydroxy-4-acetylaminobiphenyl

N-Hydroxy-2-acetylaminofluorene (N-OH-AAF), N-hydroxy-4'-fluoro-4-acetylaminobiphenyl (N-OH-FAABP) and N-hydroxy-4-acetylaminobiphenyl (N-OH-AABP) were compared for their initiation and promotion activity in the rat liver using a modified Solt-Farber system. N-OH-AAF, N-OH-FAABP and N-OH-AABP showed comparable initiation capacity when administered to male Wistar rats at a dose of 30, 120 and 120 mumol/kg respectively, 24 h after a two-thirds partial hepatectomy (PH). In contrast, only N-OH-AAF was very effective as promoter when administered to rats previously initiated with diethylnitrosamine. This was evidenced by a high number of large gamma-glutamyltranspeptidase-positive (GGT+) foci occupying a high percentage (22%) of liver volume. N-OH-FAABP was a much weaker promoter, resulting in smaller foci and lower percentage (4%) of GGT+ liver volume. The incomplete carcinogen N-OH-AABP was totally ineffective as promoter in our model. A similar difference was seen in the clastogenicity of these carcinogens in rat liver in vivo as measured by the formation of micronuclei: N-OH-AAF was far more clastogenic than N-OH-FAABP, which in turn was more clastogenic than N-OH-AABP. We have recently shown that N-acetylated deoxyguanosine adducts are responsible for clastogenicity of N-OH-AAF and may be important for promotion. DNA adduct analysis after injection of 120 mumol/kg of tritium-labeled N-OH-FAABP or N-OH-AABP, 24 h after PH, showed that N-acetylated adducts to C8 of deoxyguanosine are also formed from these structurally related liver carcinogens. However, the formation of these adducts from N-OH-FAABP and N-OH-AABP was approximately 8 and approximately 5% of the formation of dG-C8-AAF after injection of 25 mumol/kg N-OH-AAF. These data show that for the structurally related liver carcinogens N-OH-AAF, N-OH-FAABP and N-OH-AABP, clastogenicity does not predict initiating efficacy but correlates with promotion activity. Possibly, N-acetylated adducts to C8 of deoxyguanosine are involved in both clastogenicity and promotion.     

Effects of N-acetoxy-2-acetylaminofluorene and N'-nitro-N-nitrosoguanidine on nuclear DNA synthesis in rat liver.

A system for the study of DNA synthesis in isolated nuclei is described for sham and regenerating rat liver. The system has been characterized with respect to nuclear purity, conditions for optimum incorporation of [5-methyl-3H]thymidine triphosphate, time course of incorporation, product analysis by neutral and alkaline sucrose gradients, and the effect of exogenously added DNA. No difference in the basal level of activity was detected between nuclei prepared from normal or regenerating liver when isolated 24 hr after operation. However, exogenous activated DNA preferentially stimulated [5-methyl-3H]thymidine triphosphate incorporation in nuclei from regenerating liver. Activated DNA caused to react with the carcinogen N-acetoxy-2-acetylaminofluorene was a less effective primer-template in this system and decreased in a dose-dependent fashion the incorporation of [5-methyl-3H]thymidine triphosphate to below basal levels in nuclei from both normal and regenerating liver. The carcinogen N-methyl-N'-nitro-N-nitrosoguanidine had no inhibitory effect when assayed in this fashion.     

Antibodies to N-hydroxy-2-aminofluorene modified DNA as probes in the study of DNA reacted with derivatives of 2-acetylaminofluorene

Antibodies were elicited in rabbits immunized with a mixture of methylated bovine serum albumin and N-hydroxy-2-aminofluorene reacted DNA (DNA-AF). These antibodies were used in competition radioimmunoassays (RIA) with [3H]DNA-AF as radioactive antigen and different N-acetoxy-2-acetylaminofluorene (N-AcO-AAF) and N-hydroxy-2aminofluorene (N-OH-AF) reacted DNAs, deoxyguanosine, dGMP, dGpA and dApG as inhibitors. Based on the results of RIA it is concluded that the binding sites of the two residues, -AF and -AAF, to guanosine in DNA, are essentially the same. Moreover, the same sites seem to be reactive also in the direct reaction of N-AcO-AAF with dGMP. Circular dichroism measurements of modified DNA confirm the RIA results.     

Processing of 2-aminofluorene and 2-acetylaminofluorene DNA adducts in Chinese hamster ovary cells

The effects of 2-aminofluorene (AF) DNA damage on cyto-toxicityand DNA-mediated genetic transformation were investigated inChinese hamster ovary (CHO) cells. N-Acetoxy-2-acetylaminofluorene(NA-AAF) treatment of DNA repair-proficient AT3–2 cellsand UVL-10, a UV-hyper-sensitive mutant cell line derived fromAT3–2, showed that UVL-10 cells were markedly more sensitivethan AT3–2 cells to NA-AAF cytotoxicity. Analysis of cellularDNA from NA-AAF-treated cell cultures showed that AF was thepredominant DNA adduct formed in both cell lines, while formationof 2-acetylaminofluorene (AAF) DNA adducts was not detectedin cellular DNA samples of either cell line. Analysis of AFadduct removal showed that kinetics and extent of AF removalwere similar in both cell lines. The effects of cellular processingof AAF DNA adducts in CHO cells were examined by introducingplasmid pSV2gpt DNA containing AAF damage into AT3–2 andUVL-10 cell lines by transfection. For comparative purposes,AF-containing pSV2gpt was also used in parallel experiments.In transfection experiments with AAF-containing pSV2gpt DNA,yields of gpt⁺ transformants declined relative to control frequenciesin a much more pronounced manner in repair-deficient UVL-10cells than in repair-proficient AT3–2 cells. In contrast,transfection with pSV2gpt DNA containing AF adducts had no apparenteffect on transformation frequencies in either cell line, evenat very high levels of modification. Results of co-transformationexperiments in which transfected AAF-containing pSV2gpt DNAmolecules were not subjected to selection for phenotype showedthat in repair-deficient UVL-10 cells, AAF damage in pSV2gptapparently interfered with the ultimate association of transfectedDNA with recipient cell DNA.     

Prevention by thioethers of the hepatotoxicity and covalent binding to macromolecules of N-hydroxy-2-acetylaminofluorene and its sulfate ester in rat liver in vivo and in vitro

In order to find potentially effective compounds that could prevent the covalent binding of the carcinogen N-hydroxy-2-acetylaminofluorene (N-OH-AAF) to rat liver macromolecules in vivo, the prevention of the covalent binding to RNA of the sulfate ester of the carcinogen N-OH-AAF by a series of thioethers was investigated in vitro. The most effective thioethers, which inhibited the covalent binding by 70% or more, were studied for their protection against acute hepatotoxicity of N-OH-AAF in the rat in vivo. Three of these thioethers, thiazolidine, methyl 4-(methylthio)benzoate, and 2-(methylthio)benzimidazole significantly decreased the hepatoxicity of N-OH-AAF, by 45, 71 and 83%, respectively. The effects of these thioethers on the covalent binding of N-OH-AAF to cellular macromolecules in vivo were also studied. Methyl 4-(methylthio)benzoate and 2-(methylthio)benzimidazole decreased the adduct formation of N-OH-AAF to DNA by 54 and 44%, respectively, but had no effect on protein adduct formation. Only 2-(methylthio)benzimidazole caused a slight decrease (23%) in the AAF-- protein adduct formation. 2-Acetylaminofluorene (AAF) and methyl 4-(methyl-sulfinyl)benzoate were the main products in the incubation of methyl 4-(methylthio)benzoate with AAF-N-sulfate in vitro. This suggests that the thioether attacks the nitrenium ion which is formed by spontaneous breakdown of AAF-N-sulfate; the formation of a sulfonium--AAF conjugate is postulated which decomposes into AAF and a sulfinyl compound.     

Examination of genotoxicity, toxicity and morphologic alterations in hepatocytes following in vivo or in vitro exposure to methapyrilene

The antihistamine methapyrilene (MP) was widely used as a componentof cold, allergy and sleep-aid medications in the 1970s untilit was identified as a potent rat liver carcinogen. MP doesnot induce positive responses in most short-term genotoxicityassays, which suggests that it is carcinogenic by a non-genotoxicmechanism. We have evaluated the potential of MP to induce unscheduledDNA synthesis (UDS), a genetic end point and S-phase synthesis(SPS), an indicator of cell proliferation, in Fischer-344 (F344)rat and B6C3F1 mouse liver. We also examined the response ofMP in hepatocytes from two species treated in vitro. MP failedto induce UDS in rat or mouse liver following in vivo treatment,or in hepatocytes from rat and adult human treated in vitro.Control rats and mice yielded <0.3% of cells in S-phase (%S).In contrast, MP induced significant elevations in SPS both inmale F344 rat (6.3%S) and female B6C3F1 mice (1.4%S). In themale rat, sorbitol dehydrogenase (SDH), bilirubin, serum glutamicoxaloacetic transaminase (SGOT) and serum glutamic pyruvatetransaminase (SGPT) showed elevations of 9-, 10-. 17- and 28-foldover controls respectively, indicating that significant hepatotoxicitywas induced by MP. This was confirmed by histopathologic examination,which revealed significant periportal and focal necrosis followedby an increased presence of mitotic figures. These results indicatethat MP is not genotoxic in rat liver, but is a potent inducerof hepatic cell proliferation by inducing toxicity and subsequentregeneration, which may be an important mechanism of hepatocarcinogenesis.