Assessment of the genotoxicity of calcium cyclamate and cyclohexylamine

Calcium cyclamate and its major metabolite cyclohexylamine have been subjected to numerous evaluations for genetic activity. With the exception of studies for chromosome damage, the results have been negative. Results from a wide range of in vitro and in vivo cytogenetic assays ranged from clearly negative to various degrees of clastogenicity. Interpretation of the cytogenetic studies has been complicated by the conflicting responses, although some of the positive effects seem to be the consequence of secondary effects produced by high ion levels and excessive toxicity. In the studies presented here calcium cyclamate and cyclohexylamine were tested for mutagenic activity using an in vitro mammalian cells assay for gene mutation and an in vitro unscheduled DNA synthesis assay in rat hepatocytes with the Drosophila sex-linked recessive lethal assay. Calcium cyclamate was not genetically active in any of the three assays when tested to the maximum possible concentrations. The compound was largely nontoxic but did show some evidence of cytotoxicity in rat hepatocytes at concentrations of 1 mg/ml and higher. Cyclohexylamine was also negative in the three assays, but was considerably more cytotoxic at the concentrations used. The results from the three studies conducted in this evaluation are in general agreement with the majority of published genetic toxicology data for these two chemicals and indicated that the calcium cyclamate and cyclohexylamine have no direct, intrinsic genotoxicity of the type measured by these assays.     

Mutagenicity of trans,trans-muconaldehyde and its metabolites in V79 cells

Trans, trans-Muconaldehyde (MUC), a six-car-bon-diene-dialdehyde, is a microsomal, hema-totoxic ring-opened metabolite of benzene. MUC is metabolized to a variety of compounds which are formed by oxidation and/or reduction of the aldehyde group(s). In the present studies, MUC and its metabolites were examined for mutagenic activity at the hypoxanthine guanine phosphoribosyltransferase (HGPRT) locus in Chinese hamster V79 cells. Mutagenicity was scored by counting 8-azaguanine-resistant colonies. Of the 6 compounds tested, MUC and its aldehydic metabolites 6-hydroxy-trans, trans 2, 4-hexadienal and 6-oxo-trans, trans-hexadi-enoic acid were mutagenic in that order of potency. The other MUC metabolites tested (1, 6-dihydroxy-trans, trans-2, 4-hexadiene, trans, trans-muconic acid, and 6-hydroxy-trans, trans-2, 4-hexadienoic acid) had little or no activity in this system. The order of mutagenic activity of MUC and its aldehydic metabolites correlates with their reactivity towards glutathione, suggesting that alkylating potential is important in the genotoxicity of these compounds. © 1994 Wiley-Liss, Inc.     

Hypoxanthine impairs morphogenesis and enhances proliferation of a neuroblastoma model of Lesch Nyhan syndrome

Extracellular purines have essential roles in neuronal development; hence, disruptions in their metabolism as reported in Lesch Nyhan syndrome (LNS) could result in developmental abnormalities. The deficiency of hypoxanthine-guanine phosphoribosyl transferase (HGPRT) in LNS leads to increased hypoxanthine and uric acid production. We have reported that HGPRT-deficient B103-4C neuroblastoma, a neuronal model of LNS, proliferated less and differentiated more than their HGPRT-positive B103 counterparts. Here, we sought to determine whether differences in proliferation and differentiation would occur when these cells were cultured in the presence of hypoxanthine or in a hypoxanthine-/serum-free chemically defined media (NBMN2). In media with 1% serum, hypoxanthine (50 microM) significantly increased the proliferation of both cell lines with a greater effect on B103-4C cells. In 1% serum media, hypoxanthine increased differentiation of B103 but decreased B103-4C differentiation. In NBMN2, B103 proliferated far more than B103-4C, but both cell types differentiated to the same extent. These results are interpreted to suggest that elevated levels of central nervous system (CNS) hypoxanthine as reported in LNS may affect neuronal development, and to implicate hypoxanthine and abnormal neuronal development as causative factors in the etiology of LNS.     

Comparative genotoxicity evaluation of imidazolinone herbicides in somatic cells of Drosophila melanogaster.

In the present study, five analogous herbicides, namely Imazapyr (IMZR), Imazapic (IMZC), Imazethapyr (IMZT), Imazamox (IMZX) and Imazaquin (IMZQ), were evaluated for genotoxicity (mutagenic and recombinagenic activity) in the wing somatic mutation and recombination test (SMART) of Drosophila melanogaster. They are classified as imidazolinone (IMI) herbicides and their mode of action is to inhibit acetohydroxyacid synthase (AHAS), an enzyme involved in the biosynthesis of the amino acids leucine, isoleucine and valine. Two crosses were used: the standard (ST) cross and the high bioactivation (HB) cross. The latter is characterized by high levels of cytochrome P450 conferring increased sensitivity to promutagens and procarcinogens. Three-day-old larvae were exposed by chronic feeding (48 h) to four different concentrations of these herbicides (2.5, 5.0, 10.0 or 20.0 mM). For the evaluation of genotoxic effects, the frequencies of spots per individual in the treated series were compared to the concurrent negative control series (ultrapure water). Imazapyr, Imazapic and Imazethapyr gave negative results with both crosses of the wing spot test. In the ST cross, Imazamox showed positive results only for large single spots (20.0 mM IMZX) and weak positive results for total spots (10.0 and 20.0 mM IMZX), while Imazaquin showed positive results only for large single spots (5.0 and 20.0mM IMZQ) and a weak positive result for total spots (20.0 mM IMZQ). These positive results are mainly due to induced recombination and to a minor extent to mutations. In the HB cross, only Imazamox (5.0 mM IMZX) showed a weak positive result for small single spots. The positive control urethane, a promutagen, caused an increase in the number of all types of spots in both crosses. In conclusion, the results of chronic treatments performed at high doses (toxicity was observed at higher doses) shows the existence of a genotoxic risk for IMZX and IMZQ exposure under these experimental conditions, and indicate the need for further research to delineate the exact mechanisms involved.