Molecular docking of the highly hypolipidemic agent alpha-asarone with the catalytic portion of HMG-CoA reductase

Docking experiments using a number of published crystal structures of HMG-CoA reductase with the potent hypocholesterolemic agent alpha-asarone are described. The results indicate that alpha-asarone binds in the enzyme's active site. The methoxy groups play a key role in the binding and probably also in its biological activity, as shown by extensive SAR studies reported for analogues of alpha-asarone. The docking results will be valuable for the structure-based design of novel hypolipidemic agents.     

Genotoxicity assessment in aquatic environments under the influence of heavy metals and organic contaminants

The genotoxicity of river water and sediment including interstitial water was evaluated by microscreen phage-induction and Salmonella/microsome assays. Different processes used to fractionate the sediment sample were compared using solvents with different polarities. The results obtained for mutagenic activity using the Salmonella/microsome test were negative in the water and interstitial water samples analysed using the direct concentration method. The responses in the microscreen phage-induction assay showed the presence of genotoxic or indicative genotoxic activity for at least one water sample of each site analysed using the same concentration method. Similar results were obtained for interstitial water samples, i.e. absence of mutagenic activity in the Salmonella/microsome test and presence of genotoxic activity in the microscreen phage-induction assay. Metal contamination, as evidenced by the concentrations in stream sediments, may also help explain some of these genotoxic results. Stream sediment organic extracts showed frameshift mutagenic activity in the ether extract detected by Salmonella/microsome assay. The concentrates evaluated by microscreen phage-induction assay identified the action of organic compounds in the non-polar, medium polar and polar fractions. Thus, the microscreen phage-induction assay has proven to be a more appropriate methodology than the Salmonella/microsome test to analyse multiple pollutants in this ecosystem where both organic compounds and heavy metals are present.     

Genotoxicity assessment in aquatic environments under the influence of heavy metals and organic contaminants

The genotoxicity of river water and sediment including interstitial water was evaluated by microscreen phage-induction and Salmonella/microsome assays. Different processes used to fractionate the sediment sample were compared using solvents with different polarities. The results obtained for mutagenic activity using the Salmonella/microsome test were negative in the water and interstitial water samples analysed using the direct concentration method. The responses in the microscreen phage-induction assay showed the presence of genotoxic or indicative genotoxic activity for at least one water sample of each site analysed using the same concentration method. Similar results were obtained for interstitial water samples, i.e. absence of mutagenic activity in the Salmonella/microsome test and presence of genotoxic activity in the microscreen phage-induction assay. Metal contamination, as evidenced by the concentrations in stream sediments, may also help explain some of these genotoxic results. Stream sediment organic extracts showed frameshift mutagenic activity in the ether extract detected by Salmonella/microsome assay. The concentrates evaluated by microscreen phage-induction assay identified the action of organic compounds in the non-polar, medium polar and polar fractions. Thus, the microscreen phage-induction assay has proven to be a more appropriate methodology than the Salmonella/microsome test to analyse multiple pollutants in this ecosystem where both organic compounds and heavy metals are present.     

Evaluation of the genotoxicity of Crotalus durissus terrificus snake venom and its isolated toxins on human lymphocytes

In the present study, experiments were carried out to evaluate the mutagenic potential and genotoxic effects of Crotalus durissus terrificus snake venom and its isolated toxins on human lymphocytes, using the micronucleus and comet assays. Significant damage to DNA was observed for crotoxin and crotapotin (CA). Basic phospholipase A(2) (CB) and crotamine did not present any mutagenic potential when evaluated by the micronucleus test. C. d. terrificus crude venom was able to induce the formation of micronuclei, similarly to the mutagenic drug used as a positive control. In the comet assay, all the toxins tested (crotamine, crotoxin, CB and CA) and C. d. terrificus venom presented genotoxic activity. Studies on the cytogenetic toxicology of animal venoms and their isolated proteins are still very scarce in the literature, which emphasizes the importance of the present work for the identification and characterization of potential therapeutic agents, as well as for the better understanding of the mechanisms of action of toxins on the human body.     

Genotoxicity of 1,3-dichloro-2-propanol in the SOS chromotest and in the Ames test. Elucidation of the genotoxic mechanism

1,3-Dichloro-2-propanol (1,3-DCP-OH, glycerol dichlorohydrin) is of great importance in many industrial processes and has been detected in foodstuffs, in particular in soup spices and instant soups. It has been shown to be carcinogenic, genotoxic and mutagenic. Its genotoxic mechanisms are, however, not yet entirely understood. We have investigated whether alcohol dehydrogenase (ADH) catalysed activation to the highly mutagenic and carcinogenic 1,3-dichloroacetone or formation of epichlorohydrin or other genotoxic compounds play a role for mutagenicity and genotoxicity. In our studies, no indications of ADH catalysed formation of 1,3-dichloropropane could be found, although we could demonstrate a clear activation by ADH in the case of 2-chloropropenol. Formation of allyl chloride could also be excluded. We found, however, clear evidence that epichlorohydrin formed chemically in the buffer and medium used in the test is responsible for genotoxicity. No indication was found that enzymatic formation of epichlorohydrin plays a role. Additional mutagenicity and genotoxicity studies with epichlorohydrin also confirmed the hypothesis that genotoxic effects of 1,3-DCP-OH depend on the chemical formation of epichlorohydrin.     

Genotoxic activity of 1,3-butadiene and nitrogen dioxide and their photochemical reaction products in Drosophila and in the mouse bone marrow micronucleus assay.

The genotoxic activity of a photochemical reaction mixture of 1,3-butadiene and nitrogen dioxide was investigated in vivo in the mouse bone marrow micronucleus assay and the somatic mutation and recombination test in Drosophila (the wing spot test). Butadiene alone was not mutagenic in Drosophila, but induced micronuclei in mice at 10 ppm after 23 h of exposure. Nitrogen dioxide was not genotoxic in either test system. The photochemical reaction products were toxic but probably not mutagenic in Drosophila and not genotoxic in mouse bone marrow. The in vivo results do not confirm earlier in vitro results that demonstrated a strong direct-acting mutagenic activity of the photochemical products in Salmonella.     

Differential mutagenic activity of IQ (2-amino-3-methylimidazo[4,5-f]quinoline) in Salmonella typhimurium strains in vitro and in vivo, in Drosophila, and in mice

IQ, a heterocyclic aromatic amine which is formed during the frying of meat, was prepared by chemical synthesis. Its genotoxic potential was studied in bacteria, Drosophila and in mice. A mutagenic effect of IQ (frameshift induction) was detected in Salmonella typhimurium in experiments without metabolic activation; this effect was several orders of magnitude lower than that observed in the presence of an activation system. Ames tests with liver-homogenate S9 fraction from PCB-induced mice and rats confirmed the high mutagenic potency of IQ metabolites (Kasai et al., 1980a). Comparative studies on diagnostic Salmonella strains revealed that the high frameshift-inducing activity is independent of the plasmid pkM101; it is, however, greatly reduced by an intact excision-repair system for DNA lesions. The mutagenic activity of the metabolite(s) formed in vitro by S9 mix has a half-life of ca. 14 min. In the fruit fly, Drosophila melanogaster, IQ induced when used at sublethal concentrations, X-chromosomal recessive lethal mutations in male germ cells in a dose-dependent manner. In mice, tests were performed to detect somatic mutations: chromosomal anomalies (micronuclei) in bone marrow, and gene mutations (affecting coat pigmentation) in mice exposed to IQ in utero. No genotoxic effects were observed in these assays. However, the formation of mutagenic metabolites in the liver of IQ-treated mice was unequivocally demonstrated in host-mediated assays using Salmonella as mutagen probes in mice. The data demonstrate genotoxic activity of IQ in prokaryotic and eukaryotic organisms. The possible reasons for the different response of mammalian systems in vivo and the Salmonella system are discussed.     

Genotoxicity evaluation of tetramethylbenzenes

The three tetramethyl isomers of benzene (prenitene, 1,2,3,4-; izodurene, 1,2,3,5-; and durene, 1,2,4,5-tetramethylben- zene) were studied using in vitro mutagenicity and in vivo genotoxicity tests. Potency of mutate induction by these solvents was evaluated in Salmonella typhimurium cells with, and without S9-mix made from Aroclor 1254-induced rat liver S9. The potency of induction of micronuclei (MN) and sister chromatid exchanges (SCEs) by solvents was evaluated in bone marrow of mice. Izodurene displayed mutagenic potency in strains TA97a, TA98 and TA100 only in the absence of the S9-mix. In MN tests, all three tetramethylbenzenes demonstrated no clastogenic activity on the bone marrow cells. All the tested solvents were active as genotoxic compounds in the SCE tests, demonstrating a dose-response relationships.     

Evaluation of genotoxity and mutagenicity of DL-p-chlorophenylalanine, its methyl ester and some N-acyl derivatives

DL-p-chlorophenylalanine (PCPA) and its derivatives were evaluated for genotoxic effects using Escherichia coli and Bacillus subtilis strains lacking various DNA-repair mechanisms in spottest and in suspension test. The mutagenic activity of studied compounds was determined by the Ames test. Reverse mutation test was performed with Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 without S9 mix. 0.02 M nitrosomethylurea (NMU) standard mutagen was used as a positive control. The results showed that the parent nonessential amino acid PCPA had no detectable genotoxic and mutagenic activities in bacteria. The methyl ester of this amino acid and its N-phenylacetyl derivative possessed weak genotoxicity. Meanwhile N-sec-butyloxycarbonyl, N-benzyloxycarbonyl, N-(p-nitrophenylacetyl) and N-(p-nitrophenoxyacetyl) derivatives of DL-p-chlorophenylalanine exhibited appreciable genotoxicity. Among the seven tested compounds only N-benzyloxycarbonyl and N-(p-nitrophenoxyacetyl) derivatives of DL-p-chlorophenylalanine have been found to be mutagenic. Only parent PCPA possessed antimutagenic properties in respect of nitrosomethylurea. The structural modification, which strongly affects genotoxicity and mutagenicity perhaps may be due to steric hydrance of the substituents, causing interference with enzyme and DNA interactions.     

Salmonella/mammalian microsome assay with tetranitromethane and 3-nitro-L-tyrosine

The nitrosating agent tetranitromethane (TNM) and the nitrosation product 3-nitro-L-tyrosine (NT) were tested for mutagenic activity in the Salmonella/mammalian microsome assay. TNM showed strong genotoxic activity: it was mutagenic in all tester strains used (TA97, TA98, TA100, and TA102). The maximum mutagenic activity was reached between 16 and 32 micrograms/plate using the standard plate test; higher amounts led to distinct bactericidal effects. The mutagenicity was independent of an in vitro activation system. In the preincubation assay an increased bactericidal effect was observed. In contrast to TNM, NT, the nitrosation product, was non-mutagenic and non-toxic in the standard plate test and with the preincubation method up to 5000 micrograms/plate with and without S9 mix and with all tester strains used. Although TNM is a strong direct-acting mutagen, its nitrosating effect on proteins does lead to nongenotoxic nitro products of tyrosine in proteins.     

Validation of the SOS/Umu test with mutagenic complex mixtures

The SOS/Umu test, a rapid system for detecting genotoxic agents by monitoring SOS responses, was evaluated with the extracts of 8 mutagenic complex mixtures (airborne particles, coal dust, tobacco snuff, fried beef, fried shredded pork, airborne particles from polyurethane plants). In this system, the SOS function induced by genotoxicants is detected by a colorimetric measurement of beta-galactosidase in tester cells carrying a umuC-lacZ-fused gene on the plasmid. Results from the study show that a higher beta-galactosidase activity was found when the enzyme substrate and treated cells were added simultaneously into the enzyme reaction mixture and post-treatment dilution (10 X dilution with fresh medium) and incubation (for 2 h) were incorporated. The post-treatment dilution is necessary to reduce a possible false positive due to the color of test substances. The extracts of all mutagenic complex mixtures tested were found to induce dose-related SOS responses, indicating that the SOS/Umu test is potentially useful for the detection of mutagenic complex environmental mixtures.     

Genetic effects of isoniazid and the relationship to in vivo and in vitro biotransformation

The mutagenic activity of isoniazid, N-acetyl-isoniazid and hydrazine dihydrochloride was investigated in S. typhimurium. Isoniazid was found to possess a weak mutagenic activity only in repair-deficient strains TA1535 and TA100 as well as in the plasmid-containing strain TA92 (10-30 mg/plate) in the Ames test without metabolic activation. Addition of microsomal enzymes by S9 mix decreased this direct mutagenic activity. In contrast, preincubation of isoniazid with crude liver homogenate from mice, rats or Syrian golden hamsters for 4 h prior to plating with bacteria liberated a mutagenic compound which is equally active in both repair-deficient and repair wild-type strains (0.5-5 mg/plate). This activation pathway is independent of NADPH, is heat-sensitive and is operative only in a total liver homogenate in suspension. The highest capacity for mutagenic activation was achieved with liver homogenate from hamsters, followed by that from mice and rats. Furthermore, this mutagenic activation is paralleled by formation of hydrazine, as demonstrated in colorimetric measurements with p-dimethylaminobenzaldehyde. N-Acetyl-isoniazid is without mutagenic activity under similar conditions, and liberation of hydrazine was never detected. This means that, besides having a weak direct genetic activity, isoniazid is a promutagen, and formation of hydrazine is the first step in metabolic activation. It is concluded that the genotoxic properties of isoniazid in mammals are primarily determined by the pharmacokinetic behavior of the ultimate reactive metabolite. This result must be taken into consideration in risk assessment performed for mutagenic and carcinogenic properties of isoniazid in man.     

Assessment of genotoxic potential of ethylenediamine: in vitro and in vivo studies

Ethylenediamine (EDA) was evaluated for potential genotoxic activity using a battery in vitro and in vivo mammalian tests. The tests employed were the Chinese hamster ovary (CHO) gene mutation assay, the sister-chromatid exchange (SCE) test with CHO cells, unscheduled DNA synthesis (UDS) assays with primary rat hepatocytes and a dominant lethal study with Fischer 344 rats. EDA did not produce a positive, dose-related, mutagenic effect in either the CHO mutation assay or in the SCE test when evaluated both with and without the addition of a rat-liver S9 activation system. With hepatocytes, no positive effects of EDA upon UDS values were noted in 2 separate studies using either a scintillation counting procedure or an autoradiographic method to determine UDS activity. In a dominant lethal study, male rats fed for 23 weeks with dietary levels of EDA X 2HCl of 0, 0.05, 0.15 or 0.50 g/kg/day, and mated with 1 virgin female/week for 3 consecutive weeks, showed no dose-related or statistically significant effects upon fertility, total number of implantations/female, or the number of living and dead implants per female; marked effects upon the incidence of dominant lethal mutations were noted in the positive control group injected intraperitoneally with one dose of 0.25 mg/kg triethylenemelamine. We conclude that EDA was not genotoxic in the in vitro and in vivo mammalian test systems employed.     

Genotoxic evaluation of selective serotonin-reuptake inhibitors by use of the somatic mutation and recombination test in Drosophila melanogaster

This study evaluated different concentrations of selective serotonin-reuptake inhibitors (citalopram and sertraline) for genotoxicity by use of the somatic mutation and recombination test (SMART) in Drosophila melanogaster. Three-day-old larvae, trans-heterozygous for the multiple wing hairs (mwh) and flare (flr3) genes were treated with these two compounds. Two recessive markers were located on the left arm of chromosome 3, i.e. 'multiple wing hairs' (mwh) in map position 0.3 and 'flare-3' (flr3) at 38.8, while the centromere was located in position 47.7. SMART is based on the loss of heterozygosity, which may occur through various mechanisms, such as mitotic recombination, mutation, deletion, half-translocation, chromosome loss, and non-disjunction. Genetic changes occurring in somatic cells of the wing's imaginal discs, cause the formation of mutant clones on the wing blade. The results of this study show that citalopram had a genotoxic effect in the Drosophila SMART. Sertraline, however, did not show any genotoxic effect in balancer heterozygous wings. This study concluded that more information is needed to be certain regarding the mutagenic effects of sertraline.     

The role of metabolic and regulatory factors in the mutagenic activity of purine derivatives in microorganisms

The results concerning analysis of the mutagenic activity of analogues of nitrogen bases are given for Saccharomyces cerevisiae, Streptomyces antibioticus, Bacillus subtilis. The mutagenic activity of purine derivatives depends on their metabolic transformations in cell and on the activity of enzyme systems, involved in regulation of purine biosynthesis. The criterion of selection of purine analogues with genetic activity is proposed for yeasts, based on retroinhibitory ability of analogues of nitrogen bases.     

The evaluation of the genotoxicity of two commonly used food colors: Quinoline Yellow (E 104) and Brilliant Black BN (E 151)

Additives, especially colors, are in widespread use in the food industry. With the exception of the quinolines, food colors are relatively weak mutagens and are certified as safe additives despite reports that some people have allergic reactions to them. The number of food additives is still on the increase, and research on their potential mutagenic/carcinogenic activity in vivo is very expensive. Using two different cellular model systems, human lymphocytes in vitro and Vicia faba root tip meristems of in vivo, we evaluated the potential cytological and genotoxic effects of two dyes: Quinoline Yellow (E 104) and Brilliant Black BN (E 151). Two relatively new, very sensitive and rapid tests - the micronucleus and Comet assays - were used in this study. The data provided in this paper showed the genotoxic effects of the two analyzed food colors, and confirmed the diagnostic value of the MN and Comet assays for screening potentially genotoxic substances.     

Importance of multiple hydroxylated metabolites in hexamethylphosphoramide (HMPA)-mediated mutagenesis in Drosophila melanogaster

The mutagenic profiles in Drosophila and the influence of inhibition of metabolism on genotoxic activity were determined for hexamethylphosphoric triamide (HMPA), some synthetically prepared presumed metabolites and ethylated analogs. Demethylated HMPA metabolites are considerably less mutagenic than HMPA, dependent on the degree of demethylation. The mutagenicity of the presumptive primary metabolite, hydroxymethyl pentamethylphosphoramide (HM-Me5-PA), is comparable to HMPA and can be decreased considerably by inhibition of the metabolism by 1-phenylimidazole or iproniazid. This suggests that further oxidative metabolism is required for mutagenic activity. The mutagenicity of the doubly hydroxylated HMPA metabolite, N,N'-bis(hydroxymethyl)-tetramethylphosphoramide (N,N'-(HM)2-Me4-PA) can also be decreased by inhibition of metabolism, whereas the 3-fold hydroxylated N,N',-N"-(HM)3-Me3-PA is not affected by pretreatment with enzyme inhibitors, indicating that no further oxidative metabolism is required for its activation. A second hydroxylation on 1 dimethylamino group, forming N,N-(HM)2-Me4-PA, results in a drastic loss of mutagenic activity. Further oxidation of HM-Me5-PA to formyl pentamethylphosphoramide (formyl-Me5-PA) also leads to a strong reduction of the genotoxic activity. The rearrangement product of N-oxidation, N-[bis(dimethylamino)phosphinyl)-oxy)dimethylamine (HMPOA) is not mutagenic in Drosophila. The very low mutagenicity of hexaethylphosphoramide (Et6-PA) allowed us to study the mutagenicity of some ethyl-hydroxymethyl hybrid compounds. For the ethylated phosphoramides also the presence of only 1 hydroxymethyl group is insufficient for mutagenic activity, whereas the introduction of 2 or 3 hydroxymethyl groups resulted in considerable genotoxicity in the sex-linked recessive lethal (SLRL) test as well as in the ring-X loss test. It is concluded that the bioactivation of HMPA in Drosophila proceeds via multiple metabolic hydroxylations to form multifunctional, cross-linking agents. The presence of an oxygen atom on the phosphorus appears to be a prerequisite for the genotoxic activity of HMPA as hexamethylphosphorus triamide (HMPT), a derivative lacking this oxygen, is only weakly mutagenic in Drosophila. The results presented in this paper do not support the theory that formaldehyde is the active principle of activated HMPA.     

Genotoxic activity of 3-[3-phenyl-1,2,4-oxadiazol-5-yl] propionic acid and its peptidyl derivatives determined by Ames and SOS response tests

Compounds derived from 1,2,4-oxadiazole have being reported for their anti-inflamatory activity. However, those compounds should be devoid of any genotoxic side effect. In this work, the genotoxic activity of peptidomimetic moiety-containing 1,2,4-oxadiazoles derivatives was tested based on the Ames and SOS Chromotest. The results showed no mutagenic activity on the Ames test for 3-[3-phenyl-1,2,4-oxadiazol-5-yl] propionic acid (POPA) parental drug, but a weak SOS response induction on Chromotest. The chemical modifications reduced that response to a non-significative level, with l-phenylalanine peptidomimetic derivative being showing the lowest induction response. The results pointed out for the effectiveness of promoting chemical modifications of biological active compounds to increase its mode of action, showed in previous work, without increasing and even decreasing its DNA damage effect.     

Relationship between mutagenic potency in Salmonella typhimurium and chemical structure of amino- and nitro-substituted biphenyls

All positional isomers of mononitro- and monoaminobiphenyls and those of dinitro-, diamino- and aminonitrobiphenyls, which have one substituent on each benzene ring, were assayed for mutagenicity in Salmonella typhimurium by the Ames method. The results suggest that the structural requirements favoring mutagenic activity are the presence of substituents at the 4-position and their absence at the 2'-position. The introduction of an amino group to the 3'- or 4'-position of 4-nitrobiphenyl or a nitro group to 3'- or 4'-position of 4-aminobiphenyl enhanced the mutagenicity. Among the mutagenic compounds, 4-nitro analogues were mutagenic in strains TA98 and TA100 in the absence of a microsomal metabolic activation system. Strain TA98NR was not reverted by the direct-acting mutagens, whereas strain TA98/1,8-DNP6 was as revertible as strain TA98; these results suggest that the direct-acting mutagenicity involves the reduction of the nitro group by bacterial nitroreductase but does not involve specific esterification enzymes.