Architectonic distribution of the serotonin transporter within the orbitofrontal cortex of the vervet monkey.

To elucidate the organization of the serotoninergic innervation within the orbitofrontal cortex (OFC), serotonin transporter (SERT) density was quantified by autoradiography using [(3)H]cyanoimipramine binding. In six adult vervet monkeys, 15 architectonic areas were delineated according to cytoarchitectonic (Nissl), myeloarchitectonic (Gallyas) and chemoarchitectonic (acetylcholinesterase) criteria to assess SERT distribution at two levels of organization: cortical area and cortical type. For cortical type, the 15 areas were evenly divided into three different categories primarily based upon the degree of granularization of layer IV: agranular, dysgranular, and granular. Within agranular and dysgranular, but not granular cortical types, SERT density was area-specific and progressively decreased in a medial to lateral gradient. Across cortical types, SERT density decreased in a caudal to rostral gradient: agranular>dysgranular>granular. A similar caudal to rostral gradient was seen when serotonin content was measured (using high performance liquid chromatography) in areas representative of each cortical type. Collectively, these results suggest that the serotoninergic innervation is organized according to both cortical type and area, and is thus structured to differentially modulate information processing within the OFC.

Mutagenicity of hydralazine in mammalian cells and bacteria.

The genotoxicity of hydralazine (HDZ), an antihypertensive agent, was evaluated in mammalian cells and bacteria. The formation of mutants at the hypoxanthine guanine phosphoribosyl transferase locus in an adult rat liver cell line ARL 18 was determined. Bacterial mutagenicity was assessed in Salmonella typhimurium strains TA100 and TA102. The latter strain was chosen because it has A:T bases at the reversion site and HDZ has been reported to interact with thymidine. HDZ was mutagenic to ARL 18 cells with a concentration-dependent increase in mutants observed at 5 x 10(-6) to 5 x 10(-4) M. At 5 x 10(-4) M HDZ, there were 110 mutants/10(6) colony-forming cells compared to 129 for cells exposed to 10(-4) M benzo(a)pyrene, a known genotoxin. Bacterial mutants were observed with HDZ in both strains in the absence of an activating system. HDZ also induced mutants in the presence of S-9 from Aroclor-induced rat liver or uninduced rabbit liver. These results were consistent with previous reports of the mutagenicity of HDZ in TA100 and extend the observations to TA102, a strain designed to detect oxidative damage. This study also provides the first evidence of the mutagenicity of HDZ in mammalian cells. These data support the genotoxicity of HDZ in in vitro systems.

Study of potential in vitro and in vivo genotoxicity in hepatocytes of quinolone antibiotics.

The genotoxicity of quinolone antibiotics has been evaluated in hepatocytes following in vitro and in vivo exposure. Unscheduled DNA synthesis (UDS) was induced in vitro in rat hepatocytes by norfloxacin, ofloxacin, pefloxacin, and ciprofloxacin but not by nalidixic acid. In vivo UDS was not observed in hepatocytes isolated 4 to 24 hr after exposure of adult male F344 rats to either a single dose (30 to 190 mg/kg) or repeated doses (40 mg/kg) of ciprofloxacin. Using the 32P-postlabeling technique, no modified bases were detected in hepatocytes exposed in vitro to ciprofloxacin. In summary, UDS was induced in hepatocytes by in vitro exposure to high concentrations of norfloxacin, ofloxacin, pefloxacin, or ciprofloxacin. There was no evidence of in vitro DNA adduct formation by ciprofloxacin or in vivo DNA damage under the conditions tested. These findings suggest that ciprofloxacin is not DNA reactive, but it induces in vitro UDS as a consequence of some indirect action.

Evaluation of secondary nitroalkanes, their nitronates, primary nitroalkanes, nitrocarbinols, and other aliphatic nitro compounds in the Ames Salmonella assay.

The secondary nitroalkanes 2-nitropropane, 2-nitrobutane, 3-nitropentane and nitrocyclopentane, as well as their anionic forms (nitronates); the primary nitroalkanes 1-nitropropane, 1-nitrobutane, and 1-nitropentane and their respective nitronates; the nitrocarbinols 2-nitro-1-propanol, 2-nitro-1-butanol, 3-nitro-2-butanol, and 3-nitro-2-pentanol and their respective nitronates; 2-methyl-2-nitropropane, and 2-nitroso-2-nitropropane were tested in the Ames Salmonella assay using strains TA98, TA100 and TA102. Nitronates of the secondary nitroalkanes 2-nitropropane, 2-nitrobutane, 3-nitropentane, and nitrocyclopentane were significantly mutagenic in Salmonella strains TA100 and TA102 at 10-80 mumoles/plate, but the parent compounds were mutagenic at only a single dose level or were not mutagenic at all in the same dose range. The primary nitroalkanes and the nitrocarbinols were not mutagenic, or only marginally so, at the concentrations tested. The nitronates of the primary nitroalkanes and the nitrocarbinols reprotonated too rapidly under the conditions of the assay for adequate evaluation of mutagenicity. 2-Methyl-2-nitropropane was not mutagenic in strains TA100 and TA102; 2-nitroso-2-nitropropane was also not mutagenic in strains TA100 and TA102, but induced an equivocal mutagenic response in TA98. The positive Salmonella mutation data for the nitronates of the secondary nitroalkanes studied correlate very well with the very slow rate of reprotonation of secondary nitroalkane nitronates at pH 7.7 (Conaway et al. (1991) Cancer Res., 51, 3143), and provide further evidence that nitronates of secondary nitroalkanes, rather than the neutral parent forms with which they may be in equilibrium, are the more proximate mutagenic species.

Genotoxicity of fluoroquinolines and methylquinolines.

Quinoline in the presence of microsomal activation exhibits mutagenic activity in Salmonella typhimurium TA100 and induces unscheduled DNA synthesis (UDS) in rat hepatocytes. Structure-activity studies were performed to determine those positions on quinoline critically associated with its genotoxicity. In assays performed to determine the ability of 2-, 4-, 6- and 8-methylquinoline to induce UDS, only 4- and 8-methylquinoline produced a positive response. This represents an improved correlation for these methylquinolines with tumorigenic activity as compared to that previously observed with mutagenic activity in S. typhimurium TA100. The complete isomeric series of fluoroquinolines were evaluated as mutagens in S. typhimurium TA100 and for their potential to induce UDS in rat hepatocytes. The only isomers that did not exhibit significant mutagenic activity were 2- and 3-fluoroquinoline. Among these isomeric fluoroquinolines 5-, 6-, 7- and 8-fluoroquinoline are capable of inducing UDS. No significant effect on UDS was observed for 2-, 3- or 4-fluoroquinoline. These data indicate that positions 1-3 in quinoline are critical sites associated with its genotoxicity.

Sex and strain differences in the hepatocyte primary culture/DNA repair test.

The hepatocyte primary culture (HPC)/DNA repair test was developed using hepatocytes isolated from male F-344 rats. A number of genetic polymorphisms have been shown to occur in inbred strains of rats, which may lead to variation in biotransformation of xenobiotics resulting in differences in susceptibility to genotoxins. The effect of the strain utilized as a source of hepatocytes was investigated with female Lewis, F-344, and DA rats. Variation was observed when hepatocytes from the three strains were exposed to aflatoxin B1 (AFB1). Hepatocytes from female Lewis rats were more sensitive to AFB1 genotoxicity than cells from F-344 or DA rats. DNA repair was induced by a concentration as low as 10(-8) M AFB1 in the Lewis strain while 10(-4) M was needed for hepatocytes from female F-344 or DA rats. Sex-related differences were also observed with AFB1; DNA repair was induced at 10(-8) M in hepatocytes from male F-344 rats. No clearcut strain differences were seen when cells were exposed to diethylnitrosamine (DEN) or 2-acetylaminofluorene, although 10(-2) M DEN was only cytotoxic to hepatocytes from DA rats. These results demonstrate that both the strain and the sex of the animal used as a source of hepatocytes can affect the HPC/DNA repair test.

Genotoxicity of carcinogens in human hepatocytes: application in hazard assessment.

Evaluation of chemical genotoxicity has been used in assessing human cancer hazard, based on the observation that most human carcinogens are known to be DNA-reactive. The availability of data on the DNA-reactivity of compounds in metabolically competent human cells would assist hazard assessment by providing direct information of human genotoxicity. To evaluate the reliability of human hepatocytes for this purpose, the induction of DNA repair by DNA-reactive carcinogens of several structural classes and related noncarcinogens was studied. All the carcinogens elicited DNA repair synthesis, whereas the noncarcinogens did not. These studies provide additional support for the use of human hepatocytes in a DNA repair test in the investigation of genotoxicity. The demonstration of genotoxicity in human cells is suggested to provide important information for hazard assessment.

Extracellular metabolites of 2-aminofluorene in cultures of rapid and slow acetylator rabbit hepatocytes as a model for urinary and biliary metabolism.

N-acetylation is involved in determining species susceptibility to carcinogenicity by certain aromatic amines. In order to further investigate this relationship, the biotransformation of 2-aminofluorene (2-AF) by monolayer cultures of hepatocytes isolated from rapid and slow acetylator rabbits was studied. Analysis of biotransformation products liberated by cells was used as an indication of metabolites that would be excreted in the urine and bile. Hepatocytes from both acetylator phenotypes were found to extensively biotransform 2-AF. The overall rates of metabolism and the types of products formed were similar in the two phenotypes, although the quantity of several products differed. Hepatocyte cultures from rapid acetylators released a greater proportion of acetylated metabolites. Rapid acetylator hepatocytes released predominantly ring-hydroxylated-2-acetylaminofluorene (2-AAF) while the major product from the slow acetylator cultures was conjugated 2-AF. The amounts of extracellular N-hydroxy-2-acetylaminofluorene were similar in both phenotypes. No phenotype-dependent differences in extracellular metabolites were noted when hepatocytes were incubated with 2-AAF. These results indicate that hepatocytes from both phenotypes have similar capacities to excrete N-hydroxy-2-AAF and to detoxify the parent aromatic amine. These findings can be related to the carcinogenicity of 2-AF in either phenotype.

Biotransformation of aromatic amines to DNA-damaging products by urinary bladder organ cultures.

Urinary bladder is a target tissue for aromatic amine carcinogens. The intrinsic capacity of this tissue to form DNA-damaging products was investigated in explant cultures of bladder isolated from New Zealand white rabbits. DNA repair, measured by autoradiography, was used as the indicator of DNA damage. DNA repair was induced by 2-aminofluorene (2-AF) and its acetylated derivatives 2-acetyl-aminofluorene and N-hydroxy-2-acetylaminofluorene. A positive response was observed with benzidine (BZD), but no repair was seen in cultures exposed to monoacetyl BZD or diacetyl BZD. These results indicate that rabbit urinary bladder has the ability to biotransform aromatic amines to DNA-damaging products and has the capacity to repair damaged DNA. Unlike liver, where activation of BZD seems to require N-acetylation, acetylated BZD in the bladder appeared to be a detoxification product. The lack of damage by acetylated BZD is consistent with activation of BZD in bladder by prostaglandin synthetase-mediated pathways.