'Site of contact genotoxicity' assessment for implants - Potential use of single cell gel electrophoresis in biocompatibility testing of medical devices.

Toxicological risk assessment of medical devices requires genotoxicity assessment as per ISO 10993, Part 3, which is designed to address gene mutations, clastogenicity and/or aneugenicity endpoints. 'Site of contact genotoxicity' is a potential genotoxic risk especially for medical implants, that is currently not addressed in biocompatibility standards. We therefore performed initial validation study on the use of alkaline single cell gel electrophoresis (comet assay) for detecting 'site of contact genotoxicity' of medical devices, using test items made of acrylic implants impregnated with ethyl methanesulphonate (EMS). Comet assay detected increased DNA migration at the site of implantation, but not in the liver. The same implants also failed to show any genotoxicity potentials, when tested on the standard test battery using Salmonella/microsome and chromosome aberration assays. The study suggested that some medical implants can cause 'site of contact genotoxicity', without producing systemic genotoxicity. In conclusion, comet assay will add new dimension to safety assessment of medical devices, and this assay can be added to the battery of genetic toxicology tests for evaluating biocompatibility of medical implants.

Identification of Novel Molecular Regulators Modulating Ethylene Biosynthesis Using EMS-Based Genetic Screening.

The gaseous hormone ethylene regulates a diverse range of plant development and stress responses. Ethylene biosynthesis is tightly regulated by the transcriptional and posttranscriptional regulation of ethylene biosynthetic enzymes. ACC synthase (ACS) is the rate-limiting enzyme that controls the speed of ethylene biosynthesis in plant tissues, thus serving as a primary target for biotic and abiotic stresses to modulate ethylene production. Despite the critical role of ACS in ethylene biosynthesis, only a few regulatory components regulating ACS stability or ACS transcript levels have been identified and characterized. Here we show a genetic approach for identifying novel regulatory components in ethylene biosynthesis by screening EMS-mutagenized Arabidopsis seeds.

H2O2Accumulation, Host Cell Death and Differential Levels of Proteins Related to Photosynthesis, Redox Homeostasis, and Required for Viral Replication Explain the Resistance of EMS-mutagenized Cowpea to Cowpea Severe Mosaic Virus.

Infection with Cowpea severe mosaic virus (CPSMV) represents one of the main limitations for cowpea (Vigna unguiculata L. Walp.) productivity due to the severity of the disease symptoms, frequency of incidence, and difficulties in dissemination control. This study aimed to identify the proteins and metabolic pathways associated with the susceptibility and resistance of cowpea plants to CPSMV. Therefore, we treated the seeds of a naturally susceptible cowpea genotype (CE-31) with the mutagenic agent ethyl methane sulfonate (EMS) and compared the secondary leaf proteomic profile of the mutagenized resistant plants inoculated with CPSMV (MCPI plant group) to those of the naturally susceptible cowpea genotype CE-31 inoculated (CPI) and noninoculated (CPU) with CPSMV. MCPI responded to CPSMV by accumulating proteins involved in the oxidative burst, increasing H2O2 generation, promoting leaf cell death (LCD), increasing the synthesis of defense proteins, and decreasing host factors important for the establishment of CPSMV infection. In contrast, CPI accumulated several host factors that favor CPSMV infection and did not accumulate H2O2 or present LCD, which allowed CPSMV replication and systemic dissemination. Based on these results, we propose that the differential abundance of defense proteins and proteins involved in the oxidative burst, LCD, and the decrease in cowpea protein factors required for CPSMV replication are associated with the resistance trait acquired by the MCPI plant group.

Substituted cysteine scanning in D1-S6 of the sodium channel hNav1.4 alters kinetics and structural interactions of slow inactivation.

Slow inactivation in voltage-gated Na+ channels (Navs) plays an important physiological role in excitable tissues (muscle, heart, nerves) and mutations that disrupt Nav slow inactivation can result in pathophysiologies (myotonia, arrhythmias, epilepsy). While the molecular mechanisms responsible for slow inactivation remain elusive, previous studies have suggested a role for the pore-lining D1-S6 helix. The goals of this research were to determine if (1) cysteine substitutions in D1-S6 affect gating kinetics and (2) methanethiosulfonate ethylammonium (MTSEA) accessibility changes in different kinetic states. Site-directed mutagenesis in the human skeletal muscle isoform hNav1.4 was used to substitute cysteine for eleven amino acids in D1-S6 from L433 to L443. Mutants were expressed in HEK cells and recorded from with whole-cell patch clamp. All mutations affected one or more baseline kinetics of the sodium channel, including activation, fast inactivation, and slow inactivation. Substitution of cysteine (for nonpolar residues) adjacent to polar residues destabilized slow inactivation in G434C, F436C, I439C, and L441C. Cysteine substitution without adjacent polar residues enhanced slow inactivation in L438C and N440C, and disrupted possible H-bonds involving Y437:D4 S4-S5 and N440:D4-S6. MTSEA exposure in closed, fast-inactivated, or slow-inactivated states in most mutants had little-to-no effect. In I439C, MTSEA application in closed, fast-inactivated, and slow-inactivated states produced irreversible reduction in current, suggesting I439C accessibility to MTSEA in all three kinetic states. D1-S6 is important for Nav gating kinetics, stability of slow-inactivated state, structural contacts, and state-dependent positioning. However, prominent reconfiguration of D1-S6 may not occur in slow inactivation.

Migration of Q Cells in Caenorhabditis elegans.

During C. elegans larval development, the Q neuroblasts produce their lineage by three rounds of divisions along with continuous cell migrations. Their neuronal progeny is dispersed from the pharynx to the anus. This in vivo system to study cell migration is appealing for several reasons. The lineage development is stereotyped; functional analysis and genomic screens are rendered easy and powerful thanks to powerful tools; transgenic manipulations and genome engineering are efficient and can be conveniently combined with live-cell imaging. Here we describe a series of protocols in Q cell migration studies, including quantifications of progeny position, genetic screening strategies, preparation of migration mutants or transgenic worms expressing related fluorescent proteins, multipositional time-lapse tracking of Q cell migration using confocal microscopy and image analyses of single cell movements and dynamics.

New insights about the structural rearrangements required for substrate translocation in the bovine mitochondrial oxoglutarate carrier.

The oxoglutarate carrier (OGC) belongs to the mitochondrial carrier family and plays a key role in important metabolic pathways. Here, site-directed mutagenesis was used to conservatively replace lysine 122 by arginine, in order to investigate new structural rearrangements required for substrate translocation. K122R mutant was kinetically characterized, exhibiting a significant Vmax reduction with respect to the wild-type (WT) OGC, whereas Km value was unaffected, implying that this substitution does not interfere with 2-oxoglutarate binding site. Moreover, K122R mutant was more inhibited by several sulfhydryl reagents with respect to the WT OGC, suggesting that the reactivity of some cysteine residues towards these Cys-specific reagents is increased in this mutant. Different sulfhydryl reagents were employed in transport assays to test the effect of the cysteine modifications on single-cysteine OGC mutants named C184, C221, C224 (constructed in the WT background) and K122R/C184, K122R/C221, K122R/C224 (constructed in the K122R background). Cysteines 221 and 224 were more deeply influenced by some sulfhydryl reagents in the K122R background. Furthermore, the presence of 2-oxoglutarate significantly enhanced the degree of inhibition of K122R/C221, K122R/C224 and C224 activity by the sulfhydryl reagent 2-Aminoethyl methanethiosulfonate hydrobromide (MTSEA), suggesting that cysteines 221 and 224, together with K122, take part to structural rearrangements required for the transition from the c- to the m-state during substrate translocation. Our results are interpreted in the light of the homology model of BtOGC, built by using as a template the X-ray structure of the bovine ADP/ATP carrier isoform 1 (AAC1).

Scanning the effects of ethyl methanesulfonate on the whole genome of Lotus japonicus using second-generation sequencing analysis.

Genetic structure can be altered by chemical mutagenesis, which is a common method applied in molecular biology and genetics. Second-generation sequencing provides a platform to reveal base alterations occurring in the whole genome due to mutagenesis. A model legume, Lotus japonicus ecotype Miyakojima, was chemically mutated with alkylating ethyl methanesulfonate (EMS) for the scanning of DNA lesions throughout the genome. Using second-generation sequencing, two individually mutated third-generation progeny (M3, named AM and AS) were sequenced and analyzed to identify single nucleotide polymorphisms and reveal the effects of EMS on nucleotide sequences in these mutant genomes. Single-nucleotide polymorphisms were found in every 208 kb (AS) and 202 kb (AM) with a bias mutation of G/C-to-A/T changes at low percentage. Most mutations were intergenic. The mutation spectrum of the genomes was comparable in their individual chromosomes; however, each mutated genome has unique alterations, which are useful to identify causal mutations for their phenotypic changes. The data obtained demonstrate that whole genomic sequencing is applicable as a high-throughput tool to investigate genomic changes due to mutagenesis. The identification of these single-point mutations will facilitate the identification of phenotypically causative mutations in EMS-mutated germplasm.

Novel ethyl methanesulfonate (EMS)-induced null alleles of the Drosophila homolog of LRRK2 reveal a crucial role in endolysosomal functions and autophagy in vivo.

Mutations in LRRK2 cause a dominantly inherited form of Parkinson's disease (PD) and are the most common known genetic determinant of PD. Inhibitor-based therapies targeting LRRK2 have emerged as a key therapeutic strategy in PD; thus, understanding the consequences of inhibiting the normal cellular functions of this protein is vital. Despite much interest, the physiological functions of LRRK2 remain unclear. Several recent studies have linked the toxicity caused by overexpression of pathogenic mutant forms of LRRK2 to defects in the endolysosomal and autophagy pathways, raising the question of whether endogenous LRRK2 might play a role in these processes. Here, we report the characterization of multiple novel ethyl methanesulfonate (EMS)-induced nonsense alleles in the Drosophila LRRK2 homolog, lrrk. Using these alleles, we show that lrrk loss-of-function causes striking defects in the endolysosomal and autophagy pathways, including the accumulation of markedly enlarged lysosomes that are laden with undigested contents, consistent with a defect in lysosomal degradation. lrrk loss-of-function also results in the accumulation of autophagosomes, as well as the presence of enlarged early endosomes laden with mono-ubiquitylated cargo proteins, suggesting an additional defect in lysosomal substrate delivery. Interestingly, the lysosomal abnormalities in these lrrk mutants can be suppressed by a constitutively active form of the small GTPase rab9, which promotes retromer-dependent recycling from late endosomes to the Golgi. Collectively, our data provides compelling evidence of a vital role for lrrk in lysosomal function and endolysosomal membrane transport in vivo, and suggests a link between lrrk and retromer-mediated endosomal recycling.

Next-Gen Sequencing-Based Mapping and Identification of Ethyl Methanesulfonate-Induced Mutations in Arabidopsis thaliana.

Forward genetic analysis using ethyl methanesulfonate (EMS) mutagenesis has proven to be a powerful tool in biological research, but identification and cloning of causal mutations by conventional genetic mapping approaches is a painstaking process. Recent advances in next-gen sequencing have greatly invigorated the process of identifying EMS-induced mutations corresponding to a specific phenotype in model genetic hosts, including the plant Arabidopsis thaliana and the nematode Caenorhabditis elegans. Next-gen sequencing of bulked F2 mutant recombinants produces a wealth of high-resolution genetic data, provides enhanced delimitation of the genomic location of mutations, and greatly reduces hands-on time while maintaining high accuracy and reproducibility. In this unit, a detailed procedure to simultaneously map and identify EMS mutations in Arabidopsis is described.

Arabidopsis EDM2 promotes IBM1 distal polyadenylation and regulates genome DNA methylation patterns.

DNA methylation is important for the silencing of transposons and other repetitive elements in many higher eukaryotes. However, plant and mammalian genomes have evolved to contain repetitive elements near or inside their genes. How these genes are kept from being silenced by DNA methylation is not well understood. A forward genetics screen led to the identification of the putative chromatin regulator Enhanced Downy Mildew 2 (EDM2) as a cellular antisilencing factor and regulator of genome DNA methylation patterns. EDM2 contains a composite Plant Homeo Domain that recognizes both active and repressive histone methylation marks at the intronic repeat elements in genes such as the Histone 3 lysine 9 demethylase gene Increase in BONSAI Methylation 1 (IBM1) and is necessary for maintaining the expression of these genes by promoting mRNA distal polyadenylation. Because of its role in maintaining IBM1 expression, EDM2 is required for preventing CHG methylation in the bodies of thousands of genes. Our results thus increase the understanding of antisilencing, genome methylation patterns, and regulation of alternative RNA processing by intronic heterochromatin.

Genetic and chemical characterization of an EMS induced mutation in Cucumis melo CRTISO gene.

In order to broaden the available genetic variation of melon, we developed an ethyl methanesulfonate mutation library in an orange-flesh 'Charentais' type melon line that accumulates ß-carotene. One mutagenized M2 family segregated for a novel recessive trait, a yellow-orange fruit flesh ('yofI'). HPLC analysis revealed that 'yofI' accumulates pro-lycopene (tetra-cis-lycopene) as its major fruit pigment. The altered carotenoid composition of 'yofI' is associated with a significant change of the fruit aroma since cleavage of ß-carotene yields different apocarotenoids than the cleavage of pro-lycopene. Normally, pro-lycopene is further isomerized by CRTISO (carotenoid isomerase) to yield all-trans-lycopene, which is further cyclized to ß-carotene in melon fruit. Cloning and sequencing of 'yofI' CRTISO identified two mRNA sequences which lead to truncated forms of CRTISO. Sequencing of the genomic CRTISO identified an A-T transversion in 'yofI' which leads to a premature STOP codon. The early carotenoid pathway genes were up regulated in yofI fruit causing accumulation of other intermediates such as phytoene and ζ-carotene. Total carotenoid levels are only slightly increased in the mutant. Mutants accumulating pro-lycopene have been reported in both tomato and watermelon fruits, however, this is the first report of a non-lycopene accumulating fruit showing this phenomenon.

In vivo antigenotoxic potential and possible mechanism of action of selected 4-hydroxy-2H-chromen-2-one derivatives.

The in vivo sex-linked recessive lethal test was carried out in Drosophila melanogaster to investigate whether or not five substituted 4-hydroxy-2H-chromen-2-ones can modulate the genotoxicity of the well-established mutagenic agent ethyl methanesulfonate (EMS). For this purpose, 3 days old Canton S males were treated with the potent mutagen EMS alone in concentration of 0.75 ppm, as well as in combination with one of the five 4-hydroxycoumarins, namely diethyl 2-(1-(4-hydroxy-2-oxo-2H-chromen-3-yl)ethylidene)malonate (2b), 3-(1-(4-hydroxy-2-oxo-2H-chromen-3-yl)ethylidene)pentane-2,4-dione (6b), 4-(4-(4-hydroxy-2-oxo-2H-chromen-3-yl)thiazol-2-ylamino) benzenesulfonic acid (4c), 4-hydroxy-3-(2-(2-nitropheny lamino)thiazol-4-yl)-2H-chromen-2-one (9c), and (E)-4-hydroxy-3-(1-(m-tolylimino)ethyl)-2H-chromen-2-one (5d), in concentration of 70 ppm. The frequency of germinative mutations increased significantly after the treatment with EMS and decreased after treatments with coumarins. The maximum reduction was observed after treatments with 2b, 6b, 4c, and 5d. By the formation of hydrogen bonds or electrostatic interactions with O(6) of DNA guanine, tested coumarins prevent EMS-induced alkylation. The results indicate a protective role of five 4-hydroxycoumarins under the action of a strong mutagen.

The basic property of Lys385 is important for potentiation of the human α1 glycine receptor by ethanol.

Ethanol alters the function of several members of the Cys-loop ligand-gated ion channel superfamily. Recent studies have shown that the sensitivity of the α1 glycine receptor (GlyR) to ethanol can be affected by the state of G protein activation mediated by the interaction of Gßγ with intracellular amino acids in the GlyR. Here, we evaluated the physicochemical property of Lys385 that contributes to ethanol modulation by using mutagenesis, patch-clamp, and biochemical techniques. A conserved substitution (K385R) did not affect either the apparent glycine EC50 (40 ± 1 versus 41 ± 0.5 µM) or the ethanol-induced potentiation (53 ± 5 versus 46 ± 5%) of the human α1 GlyR. On the other hand, replacement of this residue with glutamic acid (K385E), an acidic amino acid, reduced the potentiation of the GlyR to 10 ± 1%. Furthermore, mutations with a hydrophobic leucine (K385L), a hydrogen bond donor glutamine (K385Q), or a neutral residue (K385A) also reduced ethanol modulation. Finally, substitution by a large and hydrophobic residue (K385F) and deletion of 385 (Lys385_) reduced ethanol modulation to 10 ± 4 and 17 ± 0.4%, respectively. Experiments using dynamic cysteine substitution with a methanethiosulfonate reagent and homology modeling indicate that the basic property and the position of Lys385, probably because of its interaction with Gßγ, is critical for ethanol potentiation of the receptor.

The ionization state of D37 in E. coli porin OmpF and the nature of conductance fluctuations in D37 mutants.

Understanding the flow of ions through E. coli porin outer membrane protein F (OmpF) requires knowledge of the charge state of all titratable residues located along the permeation pathway. Earlier theoretical studies proved successful in the calculation of the pK values of most residues. The (apparent) pK of Asp37 (D37), on the other hand, appeared rather sensitive to the (unknown) protein dielectric used. We addressed the protonation state of D37 experimentally by replacing D37 with a (neutral) valine. This D37V mutant expressed reduced cation selectivity, in agreement with the view that D37 in wild-type (WT) OmpF is fully ionized, i.e., deprotonated. The introduction of a (positively charged) arginine at position 37 evoked current fluctuations. Similar behavior was observed in the D37K mutant and the cysteine mutants D37C-MTSEA and D37C-MTSET. Nontitratable [2-(trimethylammonium)ethyl]-methanethiosulfonate (MTSET) carries a permanent and pH-independent charge of 1e, implying that the fluctuations of the D37C-MTSET mutant do not represent (de)protonation reactions of MTSET. We therefore conclude that these fluctuations reflect transitions between conformational substates evoked by structural instabilities due to the positive charge at that particular position in the pore lumen. Based on the similarities between D37C-MTSET fluctuations and those seen in the other mutants, notably D37K, the underlying mechanism of these fluctuations may be (essentially) the same in all four mutants studied.

Accessibility of the CLC-0 pore to charged methanethiosulfonate reagents.

Using the substituted-cysteine-accessibility method, we previously showed that a cysteine residue introduced to the Y512 position of CLC-0 was more rapidly modified by a negatively charged methanethiosulfonate (MTS) reagent, 2-sulfonatoethyl MTS (MTSES), than by the positively charged 2-(trimethylammonium)ethyl MTS (MTSET). This result suggests that a positive intrinsic pore potential attracts the negatively charged MTS molecule. In this study, we further test this hypothesis of a positive pore potential in CLC-0 and find that the preference for the negatively charged MTS is diminished significantly in modifying the substituted cysteine at a deeper pore position, E166. To examine this conundrum, we study the rates of MTS inhibitions of the E166C current and those of the control mutant current from E166A. The results suggest that the inhibition of E166C by intracellularly applied MTS reagents is tainted by the modification of an endogenous cysteine, C229, located at the channel's dimer interface. After this endogenous cysteine is mutated, CLC-0 resumes its preference for selecting MTSES in modifying E166C, reconfirming the idea that the pore of CLC-0 is indeed built with a positive intrinsic potential. These experiments also reveal that MTS modification of C229 can inhibit the current of CLC-0 depending on the amino acid placed at position 166.

Measurement of ethyl methanesulfonate in human plasma and breast milk samples using high-performance liquid chromatography-atmospheric pressure chemical ionization-tandem mass spectrometry.

Ethyl methanesulfonate (EMS) is a mesylate ester, which is known to be a potent mutagen, teratogen, and possibly carcinogen. Mesylate esters have been found in pharmaceuticals as contaminants formed during the manufacturing process and may potentially pose an exposure hazard to humans. We have developed and validated a method for detection of trace amounts (ng/ml levels) of EMS in human plasma and breast milk. The samples were extracted by matrix solid-phase dispersion with ethyl acetate using Hydromatrix and the ASE 200 Accelerated Solvent Extractor. The extracts were separated by high-performance liquid chromatography (HPLC) using a HILIC column. The detection was performed with a triple quadrupole mass spectrometer (TSQ Quantum Ultra, Thermo Electron Corporation) using atmospheric pressure chemical ionization in negative-ion mode and multiple reaction monitoring. The use of a surrogate internal standard in combination with HPLC-MS/MS provided a high degree of accuracy and precision. The extraction efficiency was greater than 70%. Repeated analyses of plasma and breast milk samples spiked with high (100 ng/ml), medium (50 ng/ml) and low (5 ng/ml) concentrations of the analytes gave relative standard deviations of less than 12%. The limits of detection were in the range of 0.5-0.9 ng/ml for both matrices.

Ethyl methanesulphonate in a parenteral formulation of BMS-214662 mesylate, a selective farnesyltransferase inhibitor: formation and rate of hydrolysis.

The objectives of the present study were to investigate the formation and rate of hydrolysis of ethyl methanesulphonate (EMS) in BMS-214662 mesylate drug substance and parenteral formulation by a gas chromatographic/mass spectrometric (GC/MS) method. EMS levels in the drug substance ranged between 0.3 microg/g and 0.8 microg/g. The parenteral formulation contains ethanol and the reaction between residual free methane sulphonic acid and ethanol may lead to the formation of EMS. Given that EMS is a potent mutagen, it is therefore of vital importance to eliminate or reduce the risk of human exposure. Data indicate no significant increase in the levels of EMS following storage of the drug product for 18 weeks at 25 degrees C or six weeks at 60 degrees C indicating that the potential reaction between ethanol and free methane sulphonic acid may not occur in the BMS-214662 formulation under the storage conditions evaluated and therefore causes no plausible safety concerns of EMS exposure in humans. Kinetic studies were conducted by spiking 200 ppb of EMS into water and the diluted and undiluted parenteral formulation. The rates of hydrolysis of EMS at 25 degrees C followed pseudo-first order kinetics and were determined to be 2.35 x 10(-4)min(-1), 67.4 x 10(-4)min(-1), and 1.32 x 10(-4)min(-1) in water, undiluted, and diluted drug product, respectively.

EMS in Viracept--initial ('traditional') assessment of risk to patients based on linear dose response relations.

Prior to having performed in depth toxicological, genotoxicological and DMPK studies on ethyl methanesulfonate (EMS) providing solid evidence for a thresholded dose response relationship, we had prepared and shared with regulatory authorities a preliminary risk estimate based on standard linear dose-effect projections. We estimated that maximal lifetime cancer risk was in the order of 10(-3) (for lifetime ingestion of the maximally contaminated tablets) or 10(-4) for the exposure lasting for 3 months. This estimate was based on a lifetime cancer study with methyl methanesulfonate (MMS; as insufficient data were available for EMS) in rodents and default linear back extrapolation. Analogous estimates were made specifically for breast cancer based on short term tumorigenicity studies with EMS in rats, for the induction of heritable mutations based on specific locus and dominant lethal tests in mice and for the induction of birth defects based on teratogenicity studies in mice. We concluded that even under worst case assumptions of linear dose relations the chance of experiencing these adverse effects would be very small, comprising at most a minute additional burden among the background incidence of the patients.

General 4-week toxicity study with EMS in the rat.

In this subacute toxicity study, ethyl methanesulfonate (EMS) was administered daily by oral gavage to SPF-bred Wistar rats of both sexes at dose levels of 20, 60 and 180/120 mg/kg body weight (bw)/day for a period of 28 days (for 19 days in the high-dose group). A control group was treated similarly with the vehicle, bidistilled water, only. The groups comprised 10 animals per sex, which were sacrificed after 28 days, respectively 19 days in the high-dose group, of treatment. Additional five rats per sex and group were treated accordingly and then allowed a 14 days treatment-free recovery period. Additional six rats per sex and group (three rats per sex in the control group) were treated accordingly and used for hemoglobin adduct analysis after EMS exposure. All animals survived until their scheduled necropsy. Treatment with EMS had a direct dose-dependent effect on food consumption and consequently on body weight at doses > or =20mg/kgbw/day in male rats and at > or =60 mg/kgbw/day in females rats. Hence, treatment with the high dose of 180 mg/kgbw/day had to be interrupted for 9 days after which, the animals were re-dosed at 120 mg/kgbw/day. This dose was also poorly tolerated over the remaining two treatment weeks causing again a marked reduction in food consumption and body weight. A dose of 60 mg/kgbw/day was moderately tolerated over 4 weeks treatment with mean daily food consumption and body weight distinctly lower than in controls. Primary targets of systemic toxicity were the hematopoietic system, thymolymphatic system and sexual organs. Characteristic changes in hematology parameters were decreased red blood cell counts, hematocrit, and hemoglobin concentration. White blood cell counts were also decreased due to reduced lymphocyte and granulocyte populations of each fraction. The corresponding histopathology findings were fatty atrophy of bone marrow and minimal hypocellularity of the white pulp of the spleen. Similarly, treatment with EMS caused an involution of the thymolymphatic system characterized by decreased organ weight of thymus, lymph nodes, and spleen microscopically associated with atrophy of the thymus and hypocellularity of Peyer's patches, lymph nodes and the white pulp of the spleen. The effects on sexual organs included lower organ weight/reduced size for testes, epididymides, seminal vesicles, prostate, and uterus. Tubular atrophy, single cell necrosis of the germ cells and in epididymides reduced spermatozoa were recorded microscopically. The described findings occurred at doses of 60 and 180/120 mg/kgbw/day and were dose-dependent with regard to incidence and severity. Other target organs were the pancreas (acinar cell vacuolation), thyroid gland (follicular cell hypertrophy), and salivary gland (secretory depletion of convoluted ducts). The systemic exposure to EMS was monitored by hemoglobin ethylvaline adduct measurement. The concentration of hemoglobin ethylvaline adducts was linear with the dose and accumulated 11-26-fold over the treatment period. In summary, decreases in food consumption and body weight were the dose-limiting effects of treatment with EMS. Organ toxicity was characterized by depression of cell proliferation (hematopoiesis and spermatogenesis) and changes suggestive of reduced metabolism and/or physiological imbalances (e.g. thymolymphatic system and thyroid gland) without signs of inflammatory or necrotic lesions. For some findings, especially the effects on the thymolymphatic system and sexual organs, it cannot be excluded that starvation-like condition contributed to the occurrence of such changes. The low dose of 20 mg/kgbw/day was basically free of adverse effects despite of a clear evidence for hemoglobin adducts.

Conjugal transformation and transposon and chemical mutagenesis of gram-negative selenate-respiring Citrobacter sp. strain JSA.

Conjugal mating between selenate-reducing Citrobacter sp. strain JSA and Escherichia coli S17-1 bearing pSUP2021 allowed transposon mutagenesis and chromosomal transformation. Kanamycin-resistant transconjugants were obtained successfully by this method from a freshwater selenate-respiring Citrobacter sp. strain JSA. The maximum frequency of kanamycin-resistant Tn5 transconjugants was 3.6 x 10(-6) per recipient of this strain. Of these transconjugants, eight strains of selenate reduction-deficient transconjugants living by nitrate reduction were obtained in the strain JSA. Moreover, the same phenotype of deficient mutant was created by chemical mutagenesis with ethylmethanesulfonate. The results strongly indicate that selenate reducing anaerobic respiration was independent of nitrate reduction in the Citrobacter sp. isolate strain JSA.