A toxicity evaluation and predictive system based on neural networks and wavelets.

A computational approach has been developed for performing efficient and reasonably accurate toxicity evaluation and prediction. The approach is based on computational neural networks linked to modern computational chemistry and wavelet methods. In this paper, we present details of this approach and results demonstrating its accuracy and flexibility for predicting diverse biological endpoints including metabolic processes, mode of action, and hepato- and neurotoxicity. The approach also can be used for automatic processing of microarray data to predict modes of action.

Determination of thiodyglycol in groundwater using solid-phase extraction followed by gas chromatography with mass spectrometric detection in the selected-ion mode.

A highly sensitive analytical procedure is described for determining thiodiglycol in groundwater. Samples are initially fortified with 3,3'-thiodipropanol (surrogate), then both species are extracted using sequential solid-phase extraction with both C18 and Ambersorb 572 columns. The C18 column, which removes extraneous groundwater components, is discarded; the Ambersorb 572 column is dried thoroughly before eluting polar components with a small volume of dichloromethane. The extract is taken to dryness using dry flowing nitrogen, and the resulting residue is derivatized using N-(tert.-butyldimethylsilyl)-N-methyltrifluoroacetamide and pyridine. The derivatized products are diluted to a final volume with toluene, chromatographed using a fused-silica capillary column, and detected with a quadrupole mass spectrometric detector in its selected-ion mode. Two independent, statistically unbiased, procedures were used to evaluate the detection limits for thiodiglycol; the values ranged between 4 and 16 microg(-1) groundwater.

Point mutations in the murine fumarylacetoacetate hydrolase gene: Animal models for the human genetic disorder hereditary tyrosinemia type 1.

Hereditary tyrosinemia type 1 (HT1) is a severe autosomal recessive metabolic disease associated with point mutations in the human fumarylacetoacetate hydrolase (FAH) gene that disrupt tyrosine catabolism. An acute form of HT1 results in death during the first months of life because of hepatic failure, whereas a chronic form leads to gradual development of liver disease often accompanied by renal dysfunction, childhood rickets, neurological crisis, and hepatocellular carcinoma. Mice homozygous for certain chromosome 7 deletions of the albino Tyr; c locus that also include Fah die perinatally as a result of liver dysfunction and exhibit a complex syndrome characterized by structural abnormalities and alterations in gene expression in the liver and kidney. Here we report that two independent, postnatally lethal mutations induced by N-ethyl-N-nitrosourea and mapped near Tyr are alleles of Fah. The Fah(6287SB) allele is a missense mutation in exon 6, and Fah(5961SB) is a splice mutation causing loss of exon 7, a subsequent frameshift in the resulting mRNA, and a severe reduction of Fah mRNA levels. Increased levels of the diagnostic metabolite succinylacetone in the urine of the Fah(6287SB) and Fah(5961SB) mutants indicate that these mutations cause a decrease in Fah enzymatic activity. Thus, the neonatal phenotype present in both mutants is due to a deficiency in Fah caused by a point mutation, and we propose Fah(5961SB) and Fah(6287SB) as mouse models for acute and chronic forms of human HT1, respectively.

Determination of lewisite oxide in soil using solid-phase microextraction followed by gas chromatography with flame photometric or mass spectrometric detection.

A rapid, sensitive, and convenient method is described for determining Lewisite oxide in soil. Samples are initially fortified with phenylarsine oxide (surrogate), then both species are extracted using ascorbic acid solutions containing 1,3-propanedithiol (derivatizing reagent). The corresponding filtered supernatant is sampled using a solid-phase microextraction fiber. Collected analytes are thermally desorbed in a heated gas chromatographic inlet, separated using fused-silica capillary columns ("primary" and "confirmatory"), and detected with either a mass spectrometric (selected ion monitoring mode) or flame photometric (sulfur-selective mode) detector. Two independent statistically-unbiased procedures were used to evaluate the detection limit for Lewisite oxide; the values range between 0.1 and 0.5 microg g(-1) soil.

Unscheduled DNA synthesis assay in mammalian spermatogenic cells: an update.

The unscheduled DNA synthesis (UDS) assay measures DNA repair in response to DNA damage. To date, 59 chemicals plus UV and X rays have been tested for UDS in spermatogenic cells of humans, rabbits, rats, and mice. In vivo, in vitro, and combined in vivo/in vitro procedures have been used. UDS has been shown to occur in spermatogonia, meiotic spermatocytes, and early spermatid stages. Fifty-nine percent of the agents tested gave a positive UDS response in one or more germ-cell stages. Results show 95% concordance (positive or negative) between different mammalian species. Some well-known genotoxic chemicals, for example, aflatoxin B(1) (AFB(1)), benzo[a]pyrene (B[a]P), and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), did not induce significant levels of UDS. Possible explanations are discussed. Results from the UDS assay were compared with those from the mouse specific-locus mutation (SLM) test to determine correlations between the two assays. Only two chemicals, ethyl- and methyl-nitrosourea (ENU and MNU), have been tested for UDS and SLM induction in spermatogonial stages. Results show full concordance between the two assays. In postspermatogonial stages, 25 chemicals and X rays have been tested for UDS and SLM induction. Seventy-seven percent of these agents showed similar results (positive or negative) in these germ-cell stages. Although the UDS assay cannot replace the SLM test, the strong correlations between the two assays suggest the usefulness of the UDS assay as a predictor of germ-cell mutations in mammalian systems.

Differential sensitivities of spermatogonial and postspermatogonial cell stages of the mouse to induction of unscheduled DNA synthesis by ethyl- and methyl-nitrosourea.

An autoradiographic procedure was used to measure unscheduled DNA synthesis (UDS, DNA repair synthesis) in spermatogonial and postspermatogonial cell stages of mice after treatment with two doses of N-ethyl-N-nitrosourea (ENU) and N-methyl-N-nitrosourea (MNU). Significant levels of UDS were measured in type A spermatogonia, meiotic spermatocytes, round spermatids, and early elongating spermatids but not in mature spermatids. The extent of UDS varied according to the germ cell stage and the dose. At equimolar concentrations, MNU was more efficient than ENU in eliciting a UDS response in all germ cells. After ENU treatment, type A spermatogonia showed the highest UDS response, while round and elongating spermatids showed the lowest. After MNU treatment, pachytene spermatocytes exhibited the highest UDS response while type A spermatogonia showed the lowest. The high UDS response of type A spermatogonia to ENU parallels the well-known high mutational sensitivity of spermatogonia to this chemical. Similarly, the high UDS response observed in meiotic spermatocytes and early spermatid stages after MNU treatment correlates with the high mutational sensitivity of postspermatogonial stages to MNU. Thus, the present results, like the specific locus mutation studies, indicate that ENU and MNU each has a unique effect on the spermatogenic cells. This effect is likely due to the different mechanism of action of ENU and MNU at the level of DNA and also to the physiological differences between different germ-cell stages. Teratogenesis Carcinog. Mutagen. 19:339-351, 1999. Published 1999 Wiley-Liss, Inc.

Analysis of methylphosphonic acid, ethyl methylphosphonic acid and isopropyl methylphosphonic acid at low microgram per liter levels in groundwater.

A method is described for determining methylphosphonic acid, ethyl methylphosphonic acid and isopropyl methylphosphonic acid, which are hydrolysis products of the nerve agents VX (S-2-diisopropylaminoethyl O-ethyl methylphosphonothiolate) and GB (sarin, isopropylmethyl phosphonofluoridate). The analytes are extracted from 50 ml groundwater using a solid-phase extraction column packed with 500 mg of silica with a bonded quaternary amine phase, and are eluted and derivatized with methanolic trimethylphenylammonium hydroxide. Separation and quantitation are achieved using a capillary column gas chromatograph equipped with a flame photometric detector operated in its phosphorus-selective mode. Two independent statistically-unbiased procedures were employed to determine the detection limits, which ranged between 3 and 9 micrograms/l, for the three analytes.

Dominant lethal mutations, heritable translocations, and unscheduled DNA synthesis induced in male mouse germ cells by glycidamide, a metabolite of acrylamide.

The hypothesis that acrylamide induces dominant lethal mutations and heritable translocations in male mice, not through direct adduction, but by conversion to the reactive epoxide, glycidamide, was investigated. Three studies, namely, induction of dominant lethal mutations, heritable translocations, and unscheduled DNA synthesis in spermatids, which were conducted earlier in this laboratory for acrylamide, were also performed for glycidamide to determine its mutagenic properties and to compare responses. Results of these studies are consistent with the proposal that in vivo conversion to glycidamide is responsible for the mutagenicity of acrylamide in male mice.

Acrylamide: a review of its genotoxicity and an assessment of heritable genetic risk.

An updated review of the genotoxicity studies with acrylamide is provided. Then, using data from the studies generating quantitative information concerning heritability of genetic effects, an assessment of the heritable genetic risk presented by acrylamide is presented. The review offers a discussion of the reactions and possible mechanisms of genotoxic action by acrylamide and its epoxide metabolite glycidamide. Several genetic risk approaches are discussed, including the parallelogram, direct (actually a modified direct), and doubling dose approaches. Using data from the specific-locus and heritable translocation assays, the modified direct and doubling dose approaches are utilized to quantitate genetic risk. Exposures of male parents to acrylamide via inhalation, ingestion, and dermal routes are also quantitated. With these approaches and measurements and their underlying assumptions concerning extrapolation factors (including germ cell stage specificity, DNA repair variability, locus specificity), number of human loci associated with dominant disease alleles, and spontaneous mutation rates, an assessment of heritable genetic risk for humans is calculated for the three exposure scenarios. The calculated estimates for offspring from fathers exposed to acrylamide via drinking water are up to three offspring potentially affected with induced genetic disease per 10(8) offspring. Estimates for inhalation or dermal exposures suggest higher risks for induced genetic disease in offspring from fathers exposed in occupational settings.

Induction of DNA damage by urethane in mouse testes: DNA binding and unscheduled DNA synthesis.

The extent and persistence of DNA damage and repair were investigated in mouse spermatogenic cells exposed in vivo to urethane (ethyl carbamate, EC). Adult male mice exposed to [3H]EC at 10-1,000 mg/kg were sacrificed 12 hr later. EC/metabolite binding to liver and testicular DNA and to sperm heads from the vasa deferentia was measured. Other male mice were exposed to EC at 50-750 mg/kg, and unscheduled DNA synthesis (UDS) induction was investigated in early spermatid stages. Similar experiments were conducted with vinyl carbamate (VC; putative EC metabolite) at 10-75 mg/kg. [3H]EC bound to liver and testicular DNA and to whole sperm heads. Testicular DNA binding increased linearly with dose, although binding was at least 2 orders of magnitude lower than with liver DNA. Sperm head binding also increased linearly with dose. Dose response studies with the UDS assay showed that EC and VC induced a small but significant increase of the UDS response in early spermatid stages. However, the induced UDS responses were quite variable and did not consistently increase with the administered dose. To determine the time kinetics of UDS induction, [3H]dThd was injected at various times after treatment with 500 mg/kg of EC or 60 mg/kg of VC. A slight but significant UDS increase was observed 4 hr after treatment with EC but not with VC. Overall, these results suggest that EC metabolites bind to testis DNA and cause low-level DNA damage in mouse spermatogenic cells. This type of DNA damage apparently does not have significant genetic consequences.

Ethylene oxide inhalation at different exposure-rates affects binding levels in mouse germ cells and hemoglobin. Possible explanation for the effect.

Male mice were exposed to [3H]EtO by inhalation at different exposure rates (300 parts per million (ppm) of EtO for 1 h: 150 ppm for 2 h: 75 ppm for 4 h). The total exposure was fixed at 300 ppm-h. The amount of EtO binding to developing spermatogenic stages, to sperm DNA, to testis DNA and to hemoglobin was then measured as a function of the EtO exposure rate. Generally, as the exposure rate increased there was an increase in the amount of EtO binding to the targets. For example, alkylation of sperm from the caudal epididymides 6 d posttreatment, of DNA from the vas sperm (averaged over 4 time points), of testis DNA (90 min posttreatment), and of hemoglobin (averaged over 4 time points), was 2.0 +/- 0.2 (SD), 1.8 +/- 0.4, 2.9 +/- 0.3, and 1.5 +/- 0.1 times greater, respectively, after an exposure to 300 ppm for 1 h than after an exposure to 75 ppm for 4 h. The testicular DNA from animals exposed to 300 ppm of [3H]EtO for 1 h was also analyzed for the presence of N7-hydroxyethylguanine (N7HEG) and O6-hydroxyethylguanine (O6HEG). The half-life (T1 2) of the N7HEG in the testis DNA was calculated to be 2.8 d. This lesion was removed relatively rapidly from the testis DNA and was probably excised by enzymatic repair. No formation of O6HEG was detected in any of the testis DNA samples analyzed. Additional experiments showed that the exposure rate effect was the result of less total EtO being taken in by the mice over long exposure times compared to that taken in during shorter exposure times at higher concentrations. This result argues against the idea that the exposure rate effect is the result of physiological/enzymological changes affecting transport or metabolism of the chemical within the animals under different exposure rate conditions.

Adducts in sperm protamine and DNA vs. mutation frequency.

In mammals, variability in the genetic sensitivity of different germ-cell stages to mutagens could be the result of how much chemical reaches the different stages, what molecular targets may be affected in the different stages and whether or not repair of lesions occurs. In the mouse, several chemical mutagens have been found that cause their greatest genetic damage in late-spermatid and early-spermatozoa stages and that also bind very strongly to the protamine in these stages. Chemicals which are less genetically damaging to these stages have been found to have much less affinity for protamine. Furthermore, the level of chemical binding to DNA in late-spermatid and early-spermatozoa stages has not been correlated with the level of induced genetic damage, although DNA breakage in these sensitive stages has been shown to increase. This DNA damage is believed to indirectly result from chemical binding to sulfhydryl groups in protamine which prevents normal chromatin condensation within the sperm nucleus.

Measurement of DNA breakage in specific germ-cell stages of male mice exposed to acrylamide, using an alkaline-elution procedure.

DNA breakage in spermiogenic stages of the mouse was studied after exposure to acrylamide (AA), using an alkaline-elution technique. At daily intervals over a 3-week period following i.p. injection of 100 mg AA/kg, mature spermatozoa were recovered from treated ([3H]dThd-labeled) and control ([14C]dThd-labeled) animals, and were lysed together on polycarbonate filters; the DNA was eluted with a high-pH (12.0) buffer. Elution of germ-cell DNA from AA-exposed animals increased (more DNA-strand breaks) in stages sensitive to the dominant-lethal effects of AA (late spermatids to early spermatozoa) (Shelby et al., 1986). The stage-related pattern of AA-induced DNA breakage also paralleled the pattern of sperm alkylation and protamine alkylation found to be produced by AA (Sega et al., 1989). While dominant-lethal damage from AA exposure is greatest in the spermatids and early spermatozoa, no such damage was observed in pachytene spermatocytes and early spermatids (Shelby et al., 1986). Therefore, AA-induced DNA breakage was also studied directly in pachytene spermatocytes and in early spermatids at short intervals (up to 4 days) after exposure. DNA breakage was clearly detected in these cell stages, with maximum breakage occurring at 1 day after treatment. At later times, the breakage gradually decreased, presumably as a result of DNA repair. By the time these cell stages gave rise to functional spermatozoa, DNA breaks that could have produced dominant-lethal events had apparently been reduced to a level where no genetic effect could be observed.

Review of the mutagenicity of ethylene oxide.

Ethylene oxide has been shown to be an effective mutagen in a variety of organisms ranging from bacteria to mammalian cells. There is also an association between ethylene oxide exposure and human somatic cell cytogenetic damage. Furthermore, ethylene oxide has been shown to alkylate protein and DNA at exposure levels that have been encountered occupationally. Ethylene oxide is not only effective at producing somatic cell mutations but also at inducing genetic damage in germ cells. While it is clear that ethylene oxide is a germ cell mutagen in whole mammals, the mechanism(s) by which it produces genetic lesions in germ cells is uncertain.

Acrylamide exposure induces a delayed unscheduled DNA synthesis in germ cells of male mice that is correlated with the temporal pattern of adduct formation in testis DNA.

A study of meiotic and postmeiotic germ-cell-stage sensitivity of male mice to induction of unscheduled DNA synthesis (UDS) by acrylamide showed that DNA repair could be detected in early spermatocytes (after the last scheduled DNA synthesis) through about mid-spermatid stages. No DNA repair could be detected in later stages. The maximum UDS response was observed 6 hr after i.p. exposure and was about 5 times greater than the response measured immediately after treatment. This is the longest delay between chemical treatment and maximum UDS response yet observed in mouse germ cells. There was a linear relationship between the UDS response and acrylamide exposure from 7.8 to 125 mg/kg. By using 14C-labeled acrylamide it was determined that the temporal pattern of adduct formation in testes DNA paralleled that of the UDS response, with maximum binding occurring 4 to 6 hr after exposure. In contrast, the temporal pattern of adduct formation in liver DNA showed maximum binding within 1 to 2 hr after exposure and was an order of magnitude greater than that found for the testis DNA.

Acrylamide binding to the DNA and protamine of spermiogenic stages in the mouse and its relationship to genetic damage.

Mice received an intraperitoneal injection of 14C-labeled acrylamide (AA) at an exposure of 125 mg/kg to equal that used in genetic studies carried out by Shelby et al. (1986). Subsequently, spermatozoa were recovered from the reproductive tracts of the animals over a 3-week period and assayed for the amount of bound AA. A strong increase in the level of binding occurred in late-spermatid to early-spermatozoa stages; these same stages are also genetically most sensitive to the action of AA. At all time points, alkylation of DNA within the sperm accounted for a very small fraction (generally less than 0.5%) of the total sperm-head alkylation. However, alkylation of protamine, a protein unique to sperm cells, was found to be correlated with total sperm-head alkylation and accounted for essentially all of the AA binding. Two radioactive adducts were found in hydrolysed protamine samples, one of which co-eluted with a standard of S-carboxyethylcysteine. Protamine alkylation appears to be a significant cause of acrylamide-induced genetic damage in spermiogenic cells of the mouse.

Inhalation exposure-rate of ethylene oxide affects the level of DNA breakage and unscheduled DNA synthesis in spermiogenic stages of the mouse.

The effect of ethylene oxide (EtO) inhalation-exposure rate on the induction of DNA breakage in late spermatids and on unscheduled DNA synthesis (UDS) in early spermatids was studied. The exposures were 450 parts per million (ppm) for 4 h, 900 ppm for 2 h, and 1800 ppm for 1 h. Thus, the total exposure was always 1800 ppm-h. Both DNA breakage and UDS were found to increase by a factor of approximately 3 in going from the low to high EtO concentration, suggesting that the molecular dose of EtO to the testis had increased by a similar factor. Our results are consistent with the EtO exposure-rate effect found by Generoso et al. (1986) for induction of dominant-lethal mutations in late spermatids and early spermatozoa.

Measurement of DNA breakage in spermiogenic germ-cell stages of mice exposed to ethylene oxide, using an alkaline elution procedure.

DNA breakage in spermiogenic stages of the mouse was studied after exposure to ethylene oxide (EtO), using an alkaline elution technique. At daily intervals over a 23-day period following i.p. injection of 100 mg EtO/kg, mature spermatozoa were recovered from treated ([3H]dThd-labeled) and control ([14C]dThd-labeled) animals, lysed together on polycarbonate filters, and the DNA was eluted with a high pH (12.2) buffer. Elution of germ-cell DNA from EtO-exposed animals increased (more DNA strand breaks) in stages sensitive to the genetic effects of EtO (late spermatids to early spermatozoa). The stage-related pattern of EtO-induced DNA breakage paralleled the pattern of sperm alkylation and protamine alkylation found to be produced by EtO in an earlier study (Sega and Owens, 1987). At 9 days posttreatment (sperm sampled were in late-spermatid stages at the time of EtO exposure) the amount of sperm DNA eluted did not change significantly over a pH range of 11.6-12.8, indicating that, at the time of assay, DNA breaks were already present in the sperm.

Binding of ethylene oxide in spermiogenic germ cell stages of the mouse after low-level inhalation exposure.

Mice received inhalation exposures of 3H-labeled ethylene oxide (EtO) gas at levels from 0.65 to 3.2 parts per million-hours (ppm-hr), which are below the exposure limits currently allowed for humans. Subsequently, spermatozoa were recovered from the reproductive tracts of the animals over a two-week period and assayed for the amount of bound EtO. A strong increase in the level of EtO binding occurred in late spermatid stages; these stages are also genetically sensitive to the action of EtO. The maximum binding of EtO in late spermatids amounted to 6 X 10(3) alkylations/sperm head/ppm-hr of exposure. Alkylation of the DNA within the sperm accounted for a very small fraction of the total sperm head alkylation, averaging about 20 DNA alkylations per sperm per ppm-hr of exposure over the two-week period. However, alkylation of protamine, a protein unique to sperm cells, was found to be correlated with total sperm head alkylation and accounted for nearly all of the EtO binding. Protamine alkylation appears to be a significant cause of EtO-induced genetic damage in spermiogenic cells of the mammal.