The presence of arginine may be a source of false positive results in the Ames test

An increase in the number of revertant colonies in the Ames test is generally taken as a strong indication of mutagenic activity of a test compound. However, irrelevant positive findings may constitute a major problem in regulatory drug testing. In this study, mixtures containing only amino acids such as glycine, lysine, arginine and isoleucine, routinely used as peptide preservatives in polypeptide pharmaceutical products, were investigated for mutagenesis in the Ames Salmonella typhimurium test. The results demonstrated that in the presence of metabolic activation, all the solutions containing arginine induced an increase in the number of revertant colonies in strains TA98, TA100 and TA1535 compared with the solvent control. More specifically, for strain TA98, all arginine doses tested, i.e. from 0.4 to 8 mg/plate induced a statistically significant increase in the number of revertants. This increase was biologically significant from 1.2 to 8 mg/plate. For strain TA100, the five highest test doses, i.e. from 1.2 to 8 mg/plate, induced statistically and biologically significant increases in the number of revertants. A statistically significant increase in colony number was also observed in strain TA1535, but only at the maximal test dose of 8 mg/plate arginine. These increases were observed with arginine from two different sources, suggesting that the observed effect would not be due to the presence of potential impurities in the type of arginine used. Our findings show that a functional metabolic activation system was required to induce an increase in the number of colonies. The presence of vitamin C inhibited the arginine-induced increase in the number of revertant colonies in S. typhimurium strain TA98, suggesting a potential involvement of oxidative stress.     

Predictive assay for rodent carcinogenicity using in vivo biochemical parameters: operational characteristics and complementarity.

111 chemicals of known rodent carcinogenicity (49 carcinogens, 62 noncarcinogens), including many promoters of carcinogenesis, nongenotoxic carcinogens, hepatocarcinogens, and halogenated hydrocarbons, were selected for study. The chemicals were administered by gavage in two dose levels to female Sprague-Dawley rats. The effects of these 111 chemicals on 4 biochemical assays (hepatic DNA damage by alkaline elution (DD), hepatic ornithine decarboxylase activity (ODC), serum alanine aminotransferase activity (ALT), and hepatic cytochrome P-450 content (P450)) were determined. Composite parameters are defined as follows: CP = [ODC and P450), CT = [ALT and ODC), and TS = [DD or CP or CT]. The operational characteristics of TS for predicting rodent cancer were sensitivity 55%, specificity 87%, positive predictivity 77%, negative predictivity 71%, and concordance 73%. For these chemicals, the 73% concordance of this study was superior to the concordance obtained from published data from other laboratories on the Ames test (53%), structural alerts (SA) (46%), chromosome aberrations in Chinese hamster ovary cells (ABS) (48%), cell mutation in mouse lymphoma 15178Y cells (MOLY) (52%), and sister-chromatid exchange in Chinese hamster ovary cells (SCE) (60%). The 4 in vivo biochemical assays were complementary to each other. The composite parameter TS also shows complementarity to all 5 other predictors of rodent cancer examined in this paper. For example, the Ames test alone has a concordance of only 53%. In combination with TS, the concordance is increased to 62% (Ames or TS) or to 63% (Ames and TS). For the 67 chemicals with data available for SA, the concordance for predicting rodent carcinogenicity was 47% (for SA alone), 54% (for SA or TS), and 66% (for SA and TS). These biochemical assays will be useful: (1) to predict rodent carcinogenicity per se, (2) to 'confirm' the results of short-term mutagenicity tests by the high specificity mode of the biochemical assays (the specificity and positive predictivity are both 100%), and (3) to be a component of future complementary batteries of tests for predicting rodent carcinogenicity.     

False positives in MALDI-TOF detection of ERbeta in mitochondria

Recently, Yang et al. reported that estrogen receptor beta (ERbeta) is a mitochondrial protein rather than a nuclear receptor. Because this claim would lead to a significant change in our understanding of estrogen signaling, we have attempted to reproduce the MALDI-TOF data of Yang et al. We separated proteins extracted from mouse liver mitochondria by SDS-PAGE and analysed a gel band covering the molecular weight range of 50-65 kDa by MALDI-TOF/TOF. Analysis of the data with the MASCOT database algorithm provided no evidence for the presence of ERbeta in the mitochondria. If we search (as the authors did) with only the peptide masses which match to tryptic fragments of ERbeta, ERbeta is identified with a significant score of 69. However, fragmentation of these peptides shows that they are not from ERbeta. Our conclusion is that ERbeta cannot be identified by MALDI-TOF from a mixture of mitochondrial proteins resolved on SDS-PAGE.     

Correspondence regarding Schwend and Gustafsson, "False positives in MALDI-TOF detection of ERbeta in mitochondria"

Recently, Schwend and Gustafsson tried to use the MALDI-TOF methods to confirm one of the results reported by Yang et al., which provided definitive evidences to demonstrate the localization of estrogen receptor beta (ERbeta) in the mitochondria of multiple cell types, using immunocytochemistry, immunoblot, and proteomic approaches. Analysis of the data with the MASCOT database algorithm provided no evidence for the presence of ERbeta in the mouse live mitochondria, in which very low ERbeta expression has been detected in their own report. On the other hand, our MALDI-TOF analysis using human heart mitochondrial protein has identified 7 and 8 sequences that could be potentially from ERbeta and ERbeta3, respectively, but not from ATP synthases. Further, none of the sequences identified by us as those of ERbeta and ERbeta3 shares m/z targeted by Schwend and Gustafsson in their measurements. Therefore, the claim by Gustafsson's laboratory about false positives in MALDI-TOF detection of ERbeta in mitochondria has no relevance to our report.     

Mutagenicities of 2,4- and 2,6-dinitrotoluenes and their reduced products in Salmonella typhimurium nitroreductase- and O-acetyltransferase-overproducing Ames test strains

Mutagenicities of 2,4- and 2,6-dinitrotoluene (2,4-and 2,6-DNT), and reduced metabolites formed by the incubation of 2,4- and 2,6-DNT with Salmonella typhimurium TA98, were tested using S. typhimurium YG strains possessing high level of nitroreductase (NR) and/or O-acetyltransferase (OAT) activities. All compounds tested showed greatest mutagenic activities toward strains YG1041 and YG1042, which possess high levels of NR and OAT activities. The relative mutagenic activities of 2,4-DNT and its related compounds toward YG1041 and YG1042 were aminonitrotoluenes (2A4NT, 4A2NT)<2,4-DNT<2,2′-dimethyl-5,5′-dinitroazoxybenzene (2,2′-DM-5,5′-DNAOB)4-hydroxylamino-2-nitrotoluene (4HA2NT)4,4′-dimethyl-3,3′-dinitroazoxybenzene (4,4′-DM-3,3′-DNAOB), and aminonitrotoluenes (2A4NT, 4A2NT)<2,4-DNT<4HA2NT4,4′-dimethyl-3,3′-dinitroazoxybenzene (4,4′-DM-3,3′-DNAOB)<2HA4NT, respectively. In addition, the relative mutagenic activities of 2,6-DNT and its related compounds toward YG1041 and YG1042 were 2,6-DNT<2-hydroxylamino-6-nitrotoluene (2HA6NT)<2,2′-dimethyl-3,3′-dinitroazoxybenzene (2,2′-DM-3,3′-DNAOB), and 2-amino-6-nitrotoluene (2A6NT)<2,6-DNT<2HA6NT, respectively. These results, together with previous findings, suggested that aminohydroxylamino dimethylazoxybenzenes or aminohydroxylamino dimethylazobenzenes produced either by the reduction of hydroxylaminonitrotoluenes or by the reduction of dimethyl dinitroazoxybenzenes are active metabolites responsible for the mutagenic activities of 2,4- and 2,6-DNT.     

Genotoxic potential of Cotinus coggygria Scop. (Anacardiaceae) stem extract in vivo

The intention was to evaluate the possible in vivo genotoxic potential in different cell-types, of a methanol extract obtained from the plant stem of Cotinus coggygria Scop., using the sex-linked recessive lethal (or SLRL) test and alkaline comet assay. The SLRL test, revealed the genotoxic effect of this extract in postmeiotic and premeiotic germ-cell lines. The comet assay was carried out on rat liver and bone marrow at 24 and 72 h after intraperitoneal administration. For genotoxic evaluation, three concentrations of the extract were tested, viz., 500, 1000 and 2000 mg/kg body weight (bw), based on the solubility limit of the extract in saline. Comet tail moment and total scores in the group treated with 500 mg/kg bw, 24 and 72 h after treatment, were not significantly different from the control group, whereas in the groups of animals, under the same conditions, but with 1000 and 2000 mg/kg bw of the extract, scores were statistically so. A slight decrease in the comet score and tail moment observed in all the doses in the 72 h treatment, gave to understand that DNA damage induced by Cotinus coggygria extract decreased with time. The results of both tests revealed the genotoxic effect of Cotinus coggygria under our experimental conditions.     

The mutagenic activity of ethylnitrosourea at low doses in spermatogonia of the mouse as assessed by the specific-locus test

Ethylnitrosourea is the most efficient chemical mutagen in spermatogonial stem cells of the mouse and its mutagenic activity has been intensively studied. The pertinent specific-locus mutation test results for a discussion of low dose–effect studies have been summarized and indicate: (1) A threshold dose response best characterizes the relationship between dose and mutation rate. (2) The reduced effectiveness of ethylnitrosourea in the low dose range is likely due to a saturable repair process. (3) The recovery of the saturable repair process as assessed in fractionated dose experiments is long (ca. 168 h). The dynamics of stem cell spermatogonia suggests a long time interval before the cell population passes through at least one cell division and this may be relevant to an interpretation of the fractionation effects. (4) There is a slight but important discrepancy between the predicted and observed mutagenic activity of ethylnitrosourea in the low dose range. This is interpreted to be due to the differences between a mathematical abstraction and the biological realities of the system being studied.     

Systematic characterization of high mass accuracy influence on false discovery and probability scoring in peptide mass fingerprinting

Whereas the bearing of mass measurement error on protein identification is sometimes underestimated, uncertainty in observed peptide masses unavoidably translates to ambiguity in subsequent protein identifications. Although ongoing instrumental advances continue to make high accuracy mass spectrometry (MS) increasingly accessible, many proteomics experiments are still conducted with rather large mass error tolerances. In addition, the ranking schemes of most protein identification algorithms do not include a meaningful incorporation of mass measurement error. This article provides a critical evaluation of mass error tolerance as it pertains to false positive peptide and protein associations resulting from peptide mass fingerprint (PMF) database searching. High accuracy, high resolution PMFs of several model proteins were obtained using matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FTICR-MS). Varying levels of mass accuracy were simulated by systematically modulating the mass error tolerance of the PMF query and monitoring the effect on figures of merit indicating the PMF quality. Importantly, the benefits of decreased mass error tolerance are not manifest in Mowse scores when operating at tolerances in the low parts-per-million range but become apparent with the consideration of additional metrics that are often overlooked. Furthermore, the outcomes of these experiments support the concept that false discovery is closely tied to mass measurement error in PMF analysis. Clear establishment of this relation demonstrates the need for mass error-aware protein identification routines and argues for a more prominent contribution of high accuracy mass measurement to proteomic science.     

Substitution of the use of radioactivity by fluorescence for biochemical studies of RNA

We present here the use of fluorescent methodologies for structural and functional studies of RNA in place of radioactivity. The methods are highly sensitive and quantitative with the use of an infrared fluorescence imaging system. IRD-700 and IRD-800 labels are used for fluorescence detection. Chemical probing methods are largely used for mapping RNA secondary structure and to monitor ligand interactions and conformational changes involving individual bases of RNA. The new fluorescent primer extension methodology allows simple and fast chemical probing of RNA with high sensitivity. IRD-700 and IRD-800 labeled primers can also be used to monitor protein-RNA interactions by fluorescent mobility shift assays. The speed and ease of these approaches are advantages over prior methods that used hazardous radioisotopes. Structural and biochemical investigations of RNA should benefit from the use of these fluorescent methodologies.     

Effects of PBN and OKN007 in rodent glioma models assessed by 1H MR spectroscopy

Gliomas, the most common primary brain tumors in adults, have a poor outcome. PBN (α-phenyl-tert-butylnitrone) and OKN007 (2,4-disulfophenyl-PBN) are nitrones that have demonstrated beneficial effects in many aging diseases. In this study, we evaluated the anti-tumor effects of PBN and OKN007 in several rodent glioma models (C6, RG2, and GL261) by assessing metabolite alterations with magnetic resonance spectroscopy (MRS). PBN or OKN007 was administered in drinking water before or after tumor formation. MR imaging and single-voxel point-resolved spectroscopy were done to assess tumor morphology and metabolites, after therapy. Major metabolite ratios (choline, N-acetylaspartate, and lipid (methylene or methyl), all compared to creatine), as well as quantification of individual metabolite concentrations, were assessed. Nitrones induced tumor metabolism changes that resulted in restoring major metabolite ratios close to their normal levels, in the glioma regression phase. Nitrone treatment decreased the lipid (methylene)-to-creatine ratio, as well as the estimated concentration of lipid (methylene) significantly. Alterations in lipids can be a useful marker for the evaluation of the efficacy associated with treatment and were found in this study to be related to the reduction of necrosis, but not apoptosis. OKN007 was more effective than PBN when administered after tumor formation in the C6 glioma model. In conclusion, ¹H MRS and conventional MRI are useful methods to assess and follow the response of varied glioma models to anti-tumor treatments.     

Histological mapping of biochemical changes in solid tumors by FT-IR spectral imaging

Fourier-transform infrared (FT-IR) spectral imaging was used for analyzing biochemical changes in tumor cells. Metabolic parameters of human lung A549/8 adenocarcinoma and U87 glioma cells were compared under stress conditions in culture along with tumor progression after cell implantation onto the chick embryo chorio-allantoic membrane. In cell culture, glucose consumption and lactic acid release were higher in U87 cells. A549/8 cells were less sensitive to oxidative stress as observed through changes in fatty acyl chains. In vivo biochemical mapping of highly (U87) vs. poorly (A549/8) angiogenic tumors provided results comparable to culture models. Therefore, FT-IR imaging allows detecting subtle chemical changes in tumors, which might be useful for diagnosis.     

In vivo Imaging of Optic Nerve Fiber Integrity by Contrast-Enhanced MRI in Mice

The rodent visual system encompasses retinal ganglion cells and their axons that form the optic nerve to enter thalamic and midbrain centers, and postsynaptic projections to the visual cortex. Based on its distinct anatomical structure and convenient accessibility, it has become the favored structure for studies on neuronal survival, axonal regeneration, and synaptic plasticity. Recent advancements in MR imaging have enabled the in vivo visualization of the retino-tectal part of this projection using manganese mediated contrast enhancement (MEMRI). Here, we present a MEMRI protocol for illustration of the visual projection in mice, by which resolutions of (200 µm)³ can be achieved using common 3 Tesla scanners. We demonstrate how intravitreal injection of a single dosage of 15 nmol MnCl₂ leads to a saturated enhancement of the intact projection within 24 hr. With exception of the retina, changes in signal intensity are independent of coincided visual stimulation or physiological aging. We further apply this technique to longitudinally monitor axonal degeneration in response to acute optic nerve injury, a paradigm by which Mn²⁺ transport completely arrests at the lesion site. Conversely, active Mn²⁺ transport is quantitatively proportionate to the viability, number, and electrical activity of axon fibers. For such an analysis, we exemplify Mn²⁺ transport kinetics along the visual path in a transgenic mouse model (NF-κB p50^KO) displaying spontaneous atrophy of sensory, including visual, projections. In these mice, MEMRI indicates reduced but not delayed Mn²⁺ transport as compared to wild type mice, thus revealing signs of structural and/or functional impairments by NF-κB mutations.In summary, MEMRI conveniently bridges in vivo assays and post mortem histology for the characterization of nerve fiber integrity and activity. It is highly useful for longitudinal studies on axonal degeneration and regeneration, and investigations of mutant mice for genuine or inducible phenotypes.     

The effects of maternal education versus cognitive test scores on child nutrition in Kenya

This paper estimates dynamic random effects models for intakes by dietary energy, protein, calcium, iron, zinc, niacin, riboflavin, thiamin, and vitamins A, C, D, and E 100 of Kenyan school children (6-9 years) within a multivariate longitudinal framework. The explanatory variables were socioeconomic and background variables, children's body mass index, and maternal education, cognitive test scores and morbidity spells. The model parameters are estimated using the maximum likelihood method controlling for unobserved between-children differences. The main finding is that while maternal education was usually not a significant predictor of dietary intakes, maternal scores on cognitive tests did strongly predict them. Moreover, the paternal cognitive scores and maternal morbidity levels were not significantly associated with the intakes, but an index of socioeconomic status and cash income was a significant predictor. The results indicate the need to consider broader measures of human development and of devising suitable educational programs for women without formal education.     

Analysis of biochemical variations and microRNA expression in wild (Ipomoea campanulata) and cultivated (Jacquemontia pentantha) species exposed to in vivo water stress

The current study analyses few important biochemical parameters and microRNA expression in two closely related species (wild but tolerant Ipomoea campanulata L. and cultivated but sensitive Jacquemontia pentantha Jacq.G.Don) exposed to water deficit conditions naturally occurring in the field. Under soil water deficit, both the species showed reduction in their leaf area and SLA as compared to well-watered condition. A greater decrease in chlorophyll was noticed in J. pentantha (~50 %) as compared to I. campanulata (20 %) under stress. By contrast, anthocyanin and MDA accumulation was greater in J. pentantha as compared to I. campanulata. Multiple isoforms of superoxide dismutases (SODs) with differing activities were observed under stress in these two plant species. CuZnSOD isoforms showed comparatively higher induction (~10–40 %) in I. campanulata than J. pentantha. MicroRNAs, miR398, miR319, miR395 miR172, and miR408 showed opposing expression under water deficit in these two plant species. Expression of miR156, miR168, miR171, miR172, miR393, miR319, miR396, miR397 and miR408 from either I. campanulata or J. pentantha or both demonstrated opposite pattern of expression to that of drought stressed Arabidopsis. The better tolerance of the wild species (I. campanulata) to water deficit could be attributed to lesser variations in chlorophyll and anthocyanin levels; and relatively higher levels of SODs than J. pentantha. miRNA expression was different in I. campanulata than J. pentantha.     

The interactive effect of elevated temperature on deltamethrin-induced biochemical stress responses in Channa punctata Bloch

There are reports showing interactive effect of environmental factors with the toxic outcome of chemicals. We studied the interactive effect of elevated temperature as an abiotic stressor on deltamethrin-induced biochemical stress responses in a freshwater fish, Channa punctata Bloch. Heat stress (∼12 °C above ambient temperature for 3 h) and pesticide exposure (deltamethrin 0.75 ppb for 48 h) showed significant induction of heat shock protein-70 (HSP70) in liver, kidney and gills of fishes. Elevated temperature when followed by deltamethrin exposure showed synergistic effect showing a high level of HSP70 in liver and gills whereas response in the kidney was opposite. On the contrary, when deltamethrin exposure followed the heat stress, no significant difference was observed. Protein carbonylation was found to be more pronounced in heat-stressed group compared with control fish group. A significant increase in lipid peroxidation (LPO) was observed in different tissues of fish exposed to either of the stressors. In the kidney of fish exposed to heat stress followed by deltamethrin, LPO was relatively lower as compared to other treatments. Thiols content such as reduced glutathione (GSH), total thiols (T-SH), non-protein thiols (NP-SH) and protein thiols (P-SH) showed no consistent pattern in different tissues. In deltamethrin-exposed group that was subsequently exposed to heat stress, the GSH content was higher in liver and lower in both kidney and gills when compared with other groups. Alteration in the activities of antioxidant enzymes such as catalase (CAT), glutathione S-transferase (GST), glutathione reductase (GR) and glutathione peroxidase (GPx) was also observed when fish were exposed to heat stress and/or deltamethrin. Our study demonstrated that heat stress modulated biochemical stress responses in fish showing a tissue specific pattern. This implies that fish has the capacity to elicit differential response to exposure to abiotic stressors in order to reduce the systemic magnitude of stress which may otherwise lead to severe dysfunction of vital tissues.     

Sensitivity of biochemical test in comparison with other methods for the detection of mycoplasma contamination in human and animal cell lines stored in the National Cell Bank of Iran

Mycoplasma contamination in cell culture is considered as serious problem in the manufacturing of biological products. Our goal in this research is to find the best standard and rapid method with high sensitivity, specificity, accuracy and predictive values of positive and negative results for detection of mycoplasma contamination in cell cultures of the National Cell Bank of Iran. In this study, 40 cell lines suspected to mycoplasma contamination were evaluated by three different methods: microbial culture, enzymatic mycoalert^® and molecular. Enzymatic evaluation was performed using the mycoalert^® kit while in the molecular technique, a universal primer pair was designed based on the common and fixed 16SrRNA ribosomal sequences used. Mycoplasma contaminations in cell cultures with molecular, enzymatic and microbial culture methods were determined as 57.5, 52.5 and 40 %, respectively. These results confirmed the higher rate of sensitivity, specificity and accuracy for the molecular method in comparison with enzymatic and microbial methods. Polymerase chain reaction (PCR) assay based on fixed and common sequences in the 16SrRNA, is a useful valuable and reliable technique with high sensitivity, specificity and accuracy for detection of mycoplasma contamination in cell cultures and other biological products. The enzymatic mycoalert^® method can be considered as a substitution for conventional microbial culture and DNA staining fluorochrome methods due to its higher sensitivity, specificity and speed of detection (<20 min).     

The production and modification of cytochrome P-450 difference spectra by in vivo administration of methylenedioxyphenyl compounds

The in vivo administration of piperonyl butoxide (PB) or propyl isome (PI), both methylenedioxyphenyl compounds, to mice affects the levels of several hepatic cytochrome P-450 spectral interactions. The difference spectrum produced by carbon monoxide (commonly employed as a measurement of cytochrome P-450 levels) is initially lowered in magnitude by both compounds. The reduction is followed by an increase with time indicative of cytochrome induction. This biphasic effect, when produced by piperonyl butoxide, is accompanied by specific changes in the levels of the cytochrome P-450 difference spectra produced by hexobarbital, a type-I substrate, and pyridine, a type-II substrate. In addition, the ethyl isocyanide (EtNC) equilibrium point, calculated from the pH effect on the 455-nm and 430-nm peaks of the ethyl isocyanide-cytochrome P-450 difference spectrum, undergoes a biphasic shift. In contrast, propyl isome has the same effect on the substrate difference spectra as it does on the cytochrome level and produces no change in the ethyl isocyanide equilibrium point with time.     

Predicting helical segments in proteins by a helix-coil transition theory with parameters derived from a structural database of proteins

A novel helix-coil transition theory has been developed. This new theory contains more types of interactions than similar theories developed earlier. The parameters of the models were obtained from a database of 351 nonhomologous proteins. No manual adjustment of the parameters was performed. The interaction parameters obtained in this manner were found to be physically meaningful, consistent with current understanding of helix stabilizing/destabilizing interactions. Novel insights into helix stabilizing/destabilizing interactions have also emerged from this analysis. The theory developed here worked well in sorting out helical residues from amino acid sequences. If the theory was forced to make prediction on every residue of a given amino acid sequence, its performance was the best among ten other secondary structural prediction algorithms in distinguishing helical residues from nonhelical ones. The theory worked even better if one only required it to make prediction on residues that were “predictable” (identifiable by the theory); >90% predictive reliability could be achieved. The helical residues or segments identified by the helix-coil transition theory can be used as secondary structural contraints to speed up the prediction of the three-dimensional structure of a protein by reducing the dimension of a computational protein folding problem. Possible further improvements of this helix-coil transition theory are also discussed. Proteins 28:344–359, 1997. © 1997 Wiley-Liss, Inc.     

The mutagenicity analysis of imidapril hydrochloride and its degradant,diketopiperazine derivative,nitrosation mixtures by in vitro Ames test with two strains of Salmonella typhimurium

AimThe evaluation of mutagenic properties of imidapril hydrochloride (IMD) and its degradation impurity, diketopiperazine derivative (DKP), nitrosation mixtures was conducted in order to analyze the carcinogenic risk of IMD long-term treatment in patients. In this study an in vitro Ames test with Salmonella enterica serovar Typhimurium TA 98 and TA 100 strains was used.BackgroundIMD and DKP contain nitrogen atoms, which makes them theoretically vulnerable to in vivo nitrosation with the production of N-nitroso compounds (NOC). NOC, in turn, are known animal mutagens indicating that their endogenous production from nitrosable drugs constitutes a carcinogenic hazard.Materials and methodsPure IMD sample was exposed to forced degradation conditions of increased temperature and dry air in order to achieve a DKP sample. Both samples were then treated with a nitrosating agent and the obtained nitrosation mixtures were subjected to mutagenicity analysis by the Ames test with S. typhimurium TA 98 and TA 100 strains in the presence and absence of metabolic activation system (S9 mix) using a commercial Ames MPF 98/100 microplate format mutagenicity assay kit.ResultsNone of the six concentrations of the investigated nitrosation mixtures exhibited any mutagenic potential in both S. typhimurium strains. The addition of S9 mix did not alter the non-mutagenic properties of the studied compounds.ConclusionsThe nitrite treatment of both studied compounds has no impact on their mutagenic properties under the conditions of the present studies. Hence, IMD and DKP nitrosation mixtures are classified as non-mutagens in this test.