Factors affecting R6 fungicide toxicity on sea urchin fertilization and early development: roles of exposure routes and mixture components.

A technical fungicide mixture, R6 and its components, cymoxanil (CYM) and cupric oxychloride (Cu-OCl), were tested by sea urchin bioassays (Paracentrotus lividus and Sphaerechinus granularis). A set of toxicity endpoints was evaluated including both lethal and sublethal effects with the following endpoints: (a) acute embryotoxicity, (b) developmental defects, (c) changes in sperm fertilization success, (d) transmissible damage from sperm to the offspring, and (e) cytogenetic abnormalities. Acute effects on developing embryos were observed as early (prehatch) mortality at R6 levels > or =25 microg/ml. The pesticide mixture R6 was tested at realistic concentrations, ranging from 25 ng/ ml to 2.5 microg/ml, and the two components, CYM and Cu-OCl, were tested, either alone or in mixture, at concentrations equal to their levels in the corresponding R6 solutions. R6 was either dissolved in filtered seawater (water only, W-O), or spiked in "pristine" silt-clay sediment or soil samples before bioassays. Developmental toxicity of R6, following W-O dissolution, displayed a significant dose-related increase of larval malformations and differentiation arrest at concentrations of 750 ng/ml to 2.5 microg/ml both in P. lividus and in S. granularis larvae. Developmental toxicity was removed in spiked sediment up to R6 nominal levels (25 microg/ml), 10-fold above the embryotoxic R6 levels in W-O exposure. No significant developmental toxicity was exerted by CYM or Cu-OCl (W-O exposure) up to their concentrations equivalent to 2.5 microg/ml R6. The laboratory-prepared mixture of CYM and Cu-OCl, in the same concentration range, only resulted in minor effects, as larval retardation, suggesting the presence of toxic impurities (or additional components) in the R6 formulation. When sperm from either P. lividus or S. granularis were exposed to R6 before fertilization, a W-O exposure resulted in a dose-related decrease in fertilization of P. lividus sperm (up to 250 microg/ml R6), whereas S. granularis sperm underwent a significant increase of fertilization rate at the highest R6 nominal levels (up to 25 microg/ml). Equivalent CYM or Cu-OCl levels were ineffective on sperm fertilization success in both species. The offspring of S. granularis sperm exposed to 25 microg/ml R6 showed a significant increase in larval malformations, which were not detected in the offspring of R6-exposed P. lividus sperm. Again, CYM or Cu-OCl was unable to exert any transmissible damage from sperm to the offspring in either species. The present study raises the case of possible discrepancies between toxicity of a technical mixture and of its analytical-grade components, also providing evidence for a loss of pesticide toxicity following dispersion in an environmental matrix such as sediment or soil.

The role of oxidative stress in developmental and reproductive toxicity of tamoxifen.

The antiestrogen tamoxifen (TAM) is widely used as a drug against breast cancer and is currently being tested as a chemopreventive agent. However, a number of studies showed genotoxic and carcinogenic effects of TAM. These effects are thought to be related to oxygen radical overproduction which occurs during TAM metabolic activation. There is no evidence, thus far, on TAM toxicity to embryos and gametes. The present study was designed to elucidate the mechanisms of TAM-induced developmental, reproductive and cytogenetic toxicity towards sea urchin (SU) embryos with regard to the possibility of TAM-initiated oxidative stress. Embryo cultures from SU were subjected to long-term (throughout embryogenesis) or short-term (two hours) incubation with TAM at concentrations from 10(-8) to 10(-5) M. The experiments on TAM-induced toxicity to gametes were carried out with SU sperm, or unfertilized eggs, suspended in TAM (10(-8) to 10(-6) M). To assess the effects of TAM to embryos or to gametes, developmental defects, embryonic mortality, fertilization success, and cytogenetic abnormalities were scored. Oxidative damage to DNA and lipids was detected by measurements of 8OHdG levels and lipid peroxidation, respectively. Reactive oxygen species (ROS) production by eggs and embryos was recorded by luminol-dependent chemiluminescence (LDCL) and cytochrome c reduction methods. The changes in activities of SU superoxide dismutase (SOD) and catalase were also evaluated. TAM exerted: a) early embryonic mortality to exposed embryos and to the offspring of exposed eggs; b) developmental defects to the offspring of exposed sperm; c) decrease in sperm fertilization success, and d) cytogenetic effects in the offspring of exposed sperm or eggs. These morphological effects corresponded to the state of oxidative stress in SU embryos (increased oxidative damage to DNA and lipids and induction of antioxidant enzymes). Since TAM did increase significantly ROS production by embryos, it is suggested that TAM may be metabolically activated by SU embryonic oxidases and peroxidases, which in turn could be induced by TAM. The present study provides further support to the utilization of the SU system as a useful model to help elucidate mechanisms of chemical teratogenesis and carcinogenesis.

PCR-directed DNA sequencing of "nondeletion" HPRT-mutants induced by bleomycin in CHO K1-BH4 cells.

Bleomycin is one of the radiomimetic antibiotics which induces DNA double-strand breaks by highly specific free radical attack on deoxyribose moieties in DNA. Earlier, we have shown that bleomycin induces a high proportion of large deletions involving one or more exons in the hypoxanthine-guanine phosphoribosyltransferase (hprt) locus in a Chinese hamster ovary (CHO) cell line CHO K1-BH4, in which no spontaneously occurring large deletions were detected by a polymerase chain reaction (PCR)-based deletion screening assay. Here we report the molecular nature of another class of mutants in which we did not observe any abnormal exon pattern. We refer to these mutants as the "nondeletion" type. Since bleomycin is a reactive oxygen species (ROS)-generating agent, we also studied whether the change of intracellular levels of ROS may affect the bleomycin-induced mutation spectra. We therefore also investigated the hprt mutation spectra induced by bleomycin with pretreatment by TRIEN (triethylenetetramine), a superoxide dismutase (SOD) inhibitor, and TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl), a SOD mimic. Analysis of these three bleomycin-induced "nondeletion" mutation spectra revealed that 5'-GTC-3' or 5'-GCC-3' sequences were the hot spots for single basepair deletions. Other types of mutation include abnormal cDNA or no cDNA amplification on the hprt locus. Due to the small sample size, we are unable to draw a definitive conclusion about the effects of TRIEN and TEMPOL on bleomycin-induced spectrum of "nondeletion" type hprt mutations.

Toxicological assessment of liquorice: biliary excretion in rats.

Glycyrrhizin (G) and its aglycone, glycyrrhetic acid (GA) have been prescribed for several therapeutic purposes. However, side effects have pointed out the problem of the toxicity of G. On the contrary, it was recently shown that the pure aqueous liquorice extract (LE), which also contains G, produces reduced adverse effects in rat and human, as compared to pure G, this is likely be related to differences in G bioavailability and the resulting pharmacokinetics of G and GA. Using a sensitive HPLC procedure for the determination of G and GA in rat bile, pharmacokinetics of G and GA in bile have been determined. The results of the analysis showed significantly lower concentrations of G in bile samples from rats treated with LE compared to pure G. Furthermore, LE presented a significant choleretic effect after both oral and i.v. administration, which increases the excretion rate of G. In case of GA, all the concentrations were very low, often below the detection limit. The results prompted us to assess the risk associated with liquorice intake and to determine the daily amount of pure liquorice root extract that can be safely consumed.

Cytogenetic, developmental, and biochemical effects of aluminum, iron, and their mixture in sea urchins and mussels.

The present study was undertaken to evaluate the toxicity of aluminum sulfate, ferric chloride and their 1:1 mixture (Mix) on early development, fertilization and offspring quality in three sea urchins species (Sphaerechinus granularis, Paracentrotus lividus, Psammechinus microtuberculatus) and in mussels (Mytilus galloprovincialis). The endpoints were the following: a) larval malformations; b) developmental arrest; c) embryonic mortality; d) fertilization success; e) cytogenetic effects, and f) luminol-dependent chemiluminescence (LDCL). Overall data point to the induction of developmental defects in both sea urchin and mussel embryos following exposure of embryos to Al(III) or Fe(III) (10(-7) to 10(-6) M), whereas Mix caused varied effects vs. Al(III) or Fe(III) alone, from scarce or no additive effects (M. galloprovincialis and P. lividus) to a dramatic rise in embryolethality even at nominal levels of 10(-8) M (Ps. microtuberculatus).S. granularis sperm underwent a dose-dependent decrease in fertilization success following exposure to Al(III), or Fe(III), or Mix at levels ranging from 10(-8) to 10(-5) M. A significant increase of developmental defects was observed in the offspring of S. granularis sperm exposed to micromolar levels of the agents, suggesting an Al(III)- and Fe(III)-related transmissible damage to sperm. The cytogenetic analysis of Al(III)-, Fe(III)-, or Mix-exposed S. granularis embryos showed a significant increase in mitotic aberrations. A relevant feature of the observed cytogenetic damage included scattered chromosomes, suggesting cytoskeleton damage. The LDCL emission in S. granularis embryos showed a dose-related inhibition by agent levels ranging from 10(-7) to 10(-5) M; this held true for both spontaneous and, to a larger extent, for horseradish peroxidase (HRP)-activated LDCL. LDCL associated with fertilization was affected by Al(III), Fe(III) and Mix, with a time- and dose-related shift from stimulation to inhibition. The changes observed in LDCL emission suggested that the observed damage to embryogenesis, fertilization and mitotic activity may be related, at least partly, to alterations of the embryo prooxidant state. The present data point to developmental, cytogenetic and biochemical changes related to realistic levels of Al(III), Fe(III) and their mixtures, raising concern as to their environmental, occupational and iatrogenic exposures.

Quantification, qualification, and microbial killing efficiencies of antimicrobial chlorine-based substances produced by iontophoresis.

The dependence of microbial killing on chloride ions present in solutions undergoing iontophoresis is addressed. A 400-microA current was applied to vials containing synthetic urine or saline, and the production of chlorine-based substances (CBSs) was detected by the N,N-diethyl-p-phenylene diamine colorimetric method. It was found that as the time of current application increased, the total concentration of CBSs also increased. The iontophoretic current converted (through oxidation) chloride ions present in the solutions into CBSs such as free chlorine, chlorine dioxide, chlorite, monochloramine, and dichloramine (the last two were produced by iontophoresis only when nitrogenous substances were present in the solution). Two of the CBSs (free Cl and ClO2), when they were separately added back to microbial suspensions (approximately 3 x 10(5) CFU/ml) at the same concentrations at which they were detected in either 0.46% (wt/vol) NaCl solution or synthetic urine iontophoresed for 4 h at 400 microA, reduced or eliminated bacterial genera and a fungus. However, when free Cl and ClO2 were jointly added back to microbial suspensions, bacterial and fungal killing was synergistic and more rapid and complete than when these chlorine-based biocides were added separately. Therefore, iontophoresis of solutions containing chloride ions produces chlorine-based biocides that are responsible for the antimicrobial effect of iontophoresis.

In vitro inactivation of glucose-6-phosphate dehydrogenase from human red blood cells by acrolein: a possible biomarker of exposure.

We have investigated the possibility of utilizing glucose-6-phosphate dehydrogenase (G6PD) as a macromolecular (biological) marker of acrolein exposure. The result showed a dose-dependent inactivation of the erythrocyte G6PD in situ or as a purified enzyme from human erythrocytes or yeast. Amino acid analysis on the chemically modified yeast G6PD showed a formation of a lysine adduct which is probably linked to the inactivation.

Prediction of toxicological interactions in a binary mixture by using pattern recognition techniques: proposed approach with a developed model.

A model has been suggested to predict the nature of toxicological interaction in binary mixtures. The approach uses the NLM-HSDB computerized data bases, the ATSDR toxicologic profiles, and other literature for categorizing the nature (synergistic, antagonistic or no interaction) and degree of interaction. Multivariate modeling (pattern recognition techniques) is the statistical approach utilized to separate groups of compounds into those that interact synergistically or antagonistically with a given toxic compound. Preliminary results indicate 1) that there are sufficient data in the literature on interactions to permit such modeling and 2) that in the case of carbon tetrachloride those compounds that interact synergistically with it are more similar to each other than those that interact antagonistically with respect to a number of structural and toxicologic parameters. This suggested approach of utilizing pattern recognition tools will be quite useful for regulatory agencies in predicting toxicological interactions occurring in complex chemical mixtures in the environment.

Aromatic amines and acetamides in Salmonella typhimurium TA98 and TA100: a quantitative structure-activity relation study.

The mutagenicity of a series of 19 aromatic amines had been previously measured in Salmonella typhimurium strains TA98 (frame-shift) and TA100 (base-pair) with the addition of S9 from Aroclor 1254-induced rat liver. A quantitative structure-activity relation (QSAR) study using multiple regression analysis points out the influence of three factors on mutagenicity: lipophilic character, position of the amine group, and whether it is free or acetylated, as expressed by log P and two indicator variables I1 and I2, respectively. The multiple regression equations explain 78 and 88% of the variance in log mutagenicity in TA98 and TA100, respectively. First of all, mutagenicity was shown to increase with lipophilicity. On the other hand, mutagenicity is reduced when the amine or acetamido position is ortho to the juncture because of steric hindrance in its biotransformation compared with a non-ortho isomer. It is decreased also by the acetylation of the amine group, probably because the acetyl group needs to be first split off prior to oxidation of the amine group to -NHOH.

Effects of sodium azide on sea urchin embryos and gametes.

Sodium azide (SA) was tested on sea urchin embryos and gametes (Paracentrotus lividus). Developing embryos were exposed to SA (10(-6) to 10(-3) M) up to pluteus larval stage, or for shorter intervals before or after hatching. Developmental defects in SA-exposed embryos consisted mainly of gut abnormalities, without any detectable differences between pre- or post-hatch-exposed embryos. SA-induced damage to gut was exerted during gastrulation, as evident by lectin binding of extracellular matrix. No mitotic damage was observed in SA-exposed embryos, nor could pH-related variations be detected in SA-induced embryotoxicity at pH's ranging from 8 to 6. Concurrently, no effect ensued in the exposure of unfertilized eggs to SA (10(-5) to 10(-2) M) both in terms of fertilization success and of offspring quality. When sperm were suspended in filtered seawater at pH's ranging from 8 to 6, and SA levels ranging from 10(-5) to 10(-2) M, fertilization success of SA-exposed sperm appeared to be modulated by pH, by displaying three distinct dose-response trends at pH 8, 7, or 6. The consequences of sperm pretreatment on offspring quality failed to show any significant SA-induced changes on larval malformations or mortality, while confirming the previously reported pH-induced increase of developmental defects in the offspring of acid-exposed sperm (Pagano et al.: Teratogenesis Carcinogen Mutagen 5:113-121, 1985).

Effect of mercuric ion on attraction to light of artemia sp nauplii.

Living organisms exhibit a phototactic response which can be altered by certain environmental toxic chemical species. The analysis of photobehavior can help in elucidating environmental factors that influence photomotility reactions of the organisms. A method has been developed that measures the phototactic response of Artemia nauplii under the influence of mercuric ion (Hg2+) in synthetic seawater. The phototactic response of Artemia nauplii was manifested by movement of the organisms from a darkened half to lighted half of an experimental vessel containing synthetic seawater. The density as a function of time of Artemia nauplii is determined by removing aliquots from both light and dark sides and then plating on agar for counting under the dissecting microscope. Measurements consistently show a significant movement of nauplii to the lighted side within 45 min of the start of the experiments. The present investigation demonstrated that at concentrations as low as 0.010 mg HgCl2/liter there is an enhancement of phototactic effect on Artemia nauplii by mercuric ion as compared with control. The phototactic response of Artema nauplii is altered by mercuric ion in a dose-related manner, but the mechanism of this effect is presently unknown.

Effect of acute acrylonitrile exposure on metrazol induced seizures in the rat.

The effects of acute exposure to acrylonitrile (ACN), 10, 20, or 40 mg/kg by gavage, on the ability of metrazol (MTZ) to induce seizures was studied in adult, male Sprague-Dawley rats. The frequency of seizure occurrence and the frequency of a lethal seizure was greater when the high ACN dosage was given in combination with metrazol. This dosage of ACN was not lethal when given alone. Examination of brain tissue in these animals revealed no difference in cyanide levels when MTZ was combined with ACN. However, brain cytochrome c was significantly lower in animals given ACN+MTZ and brain cholinesterase was significantly higher. These results suggest that the enhanced lethality occurring in animals exposed to the combination of ACN+MTZ is not due to cyanide, a metabolic product of ACN, but rather to a potentiation of other effects of ACN perhaps involving cholinergic neurotransmission.

Occupational exposure to formaldehyde in a medical center autopsy service.

The formaldehyde exposures occurring in the autopsy service of a medical complex were evaluated as part of a study to detect genetically harmful effects of chemical exposures. Determination of time-weighted average (TWA) exposures and characterization of the patterns of exposure experienced by individuals with different work responsibilities in this occupational setting were sought. Both general area and breathing zone samples were evaluated. Estimated weekly time-weighted average exposures for pathologists, residents and technicians were determined to be between 0.61 and 1.32 parts per million with little difference between work roles. While the averages were similar, the patterns of exposure of technicians and physicians were different. Technicians were exposed to a baseline level of formaldehyde for a prolonged period of time. In contrast, physicians were exposed for shorter times but experienced higher levels during specific tasks, particularly tissue-sectioning and examination. Evaluations of work procedures and environmental conditions in autopsy services are recommended to reduce personnel exposure to formaldehyde vapor.

The evaluation of mutagenicities of 19 structurally related aromatic amines and acetamides in Salmonella typhimurium TA98 and TA100.

19 aromatic amines were assayed for mutagenicity using Salmonella typhimurium strains TA98 and TA100 with and without the addition of S9 from Aroclor-1254-induced rat liver. These included: naphthalenes (1-amino-, 1-acetamido-, 2-amino-, 2-acetamido-, 1-amino-4-nitro- and 2-amino-1-nitro-), biphenyls (2-amino-, 2-acetamido-, 4-amino- and 4-acetamido-), fluorenes (2-amino- and 2-acetamido-), anthracenes (1-amino-, 1-acetamido-, 2-amino- and 2-acetamido-), 3-aminofluoranthene, 1-aminopyrene and 6-aminochrysene. None of the compounds were mutagenic when tested without S9. With S9, 15 of 19 were mutagenic for TA98 and 16 of the 19 were mutagenic for TA100. Overall, 2-aminoanthracene was the most potent mutagen. When compared to the parent amines, the respective acetamido derivatives were consistently less mutagenic.

Transient decrease of liver cytosolic glutathione S-transferase activities in rats given 1,2-dibromoethane or CCl4.

In vivo treatment of fasted male rats with 1,2-dibromoethane (DBE) (0.4 mmol/kg) or carbon tetrachloride (CCl4) (4 mmol/kg) was found to rapidly alter the activities of liver cytosolic and microsomal glutathione S-transferases. Microsomal activities towards chloro-2,4-dinitrobenzene (CDNB) were increased 2 h after either treatment. Cytosolic activities towards CDNB and 3,4-dichloronitrobenzene (DCNB), but not 1,2-epoxy-3-(p-nitrophenoxy)-propane (ENPP), were selectively and transiently decreased after either treatment. Time course studies in DBE animals indicated that the decrease in cytosolic activity was not evident until 2 h although liver glutathione (GSH) concentrations were diminished within 15 min. In contrast, in CCl4 animals the decrease in cytosolic activity was evident within 15 min and was not accompanied by diminished GSH concentrations. By 4 h, cytosolic activities had rebounded to control levels in both DBE and CCl4-treated animals. Kinetic studies of the enzyme in liver cytosol from animals 2 h after treatment with DBE or CCl4 indicated that both treatments decreased the apparent Vmax while neither treatment altered the apparent Km. This pattern of change allows exclusion of a simple competitive mechanism of enzyme inhibition, but cannot distinguish between reversible non-competitive inhibition and irreversible inhibition. It is possible that the observed decreases in the activities of the abundant cytosal enzyme are due to 'sacrificial' covalent linkages between the enzyme and reactive metabolites of DBE or CCl4.