Gary A. Sega

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.

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.

A study of unscheduled DNA synthesis induced by X-rays in the germ cells of male mice.

Abstract • In vivo DNA repair occurring in meiotic and postmeiotic germ-cell stages of male mice after X-ray treatment has been studied. Making use of the fact that no scheduled DNA synthesis occurs in the germ cells after the last S period in primary spermatocytes, and administering testicular injections of [ 3 H]dt, we measured DNA repair after X-ray exposure by the unscheduled incorporation of [ 3 H]dT (UDS) into germ cells. • An exposure—response study showed that UDS induced in spermatids is an increasing function of X-ray exposure, at least up to 1000 R. A significant level of UDS was measured in the spermatids at an exposure level of 200 R, which is considerably lower than what has generally been required to measure UDS in mammalian cells. • When testicular injections of [ 3 H]dT were administered immediately before X-ray teatment, the amount of unscheduled incorporation of [ 3 H]dT into spermatid DNA was maximized. By 4 h following X-ray exposure, no UDS could be detected in the spermatids. This is in marked contrast to the UDS induced in spermatids by alkylating agents such as MMS, EMS, PMS, and IMS, which can still be detected at least 3 days after chemical treatment. In addition, the total amount of UDS measured in spermatids after X-ray treatment is much less than that observed after exposures to these alkylating agents which produce equivalent genetic effects. This may result from relatively fewer repairable DNA lesions being induced by X-rays and from differences in the sizes of the repaired regions. UDS induced after X-ray treatment was detected in the same germ-cell stages (early meiosis through midspermatids) that undergo UDS when exposed to EMS. However, unlike EMS, which produces about the same level of UDS in all the germ-cell stages capable of repair, X-rays appear to produce a higher level of UDS in the earlier meiotic stages. • From genetic data obtained by other workers using X-rays we have observed that dominant-lethal frequencies are at least twice as high in germ-cell stages undergoing UDS than in later stages where no UDS is detected. Furthermore, specific-locus mutation frequencies for germ-cell stages that undergo UDS after a 300-R X-ray exposure are no lower than in later stages where no UDS is observed. Although UDS has been found to occur in germ-cell stages of male mice from early meiosis to midspermatids after X-ray treatment, there is no evidence that this UDS results in a decrease of genetic damage in the germ cells in which it occurs.

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.

Dosimetry studies on the ethylation of mouse sperm DNA after in vivo exposure to [3H]ethyl methanesulfonate ☆

Abstract Methods for determining the chemical dose of ethyl methanesulfonate (EMS) to the DNA of mouse spermatozoa in the vasa deferentia and epididymides have been developed. These include procedures for the removal of contaminating protamine, which, like DNA, possesses nucleophilic sites that can be ethylated by EMS. At least 99% of all sperm protamine (at a 95% confidence level), as well as any other cellular contaminants, is removed during purification of the DNA. The purified DNA recovered from spermatozoa gives no indication of a preferential recovery of either (G+C)-rich or (A+T)-rich regions of the mouse genome: the [ 14 C]dT/[ 3 H]dC ratios for whole sperm and sperm DNA were the same for each animal tested. The spermatozoa of males used in the dosimetry studies were labeled with [ 14 C]thymidine, and then the animals were given various [ 3 H]EMS doses intraperitoneally. A constant exposure time of 4 h was used. The ratios of 3 H and 14 C activities in whole sperm and purified sperm DNA were used to measure the percentage of the total sperm ethylation occurring in the DNA. The maximum percentage found was about 18% in the dose range of 100–400 mg/kg. Values for the ethylations per nucleotide ( E / N ) ranged from ∼ 10 −7 at 3.3 mg/kg up to ∼ 10 −4 at 400 mg/kg, and the data indicated that E / N increased with the 1.5 power of the dose. E / N was also measured in testicular DNA, and the values obtained were close to those found for spermatozoan DNA. The results of such chemical dosimetry studies will be far-reaching in the interpretation of molecular events responsible for genetic alterations. As an example, dominant lethal studies by others, using EMS in the dose range considered in the present paper, have shown little or no effect until two or more days after injection of the mutagen into male mice. Since many sperm DNA ethylations are found after a 4-h exposure to EMS it appears that most of these DNA ethylations are not genetically important, at least in the production of dominant lethals, and that perhaps genetic damage occurs only at rarely ethylated DNA sites.

Methylation of DNA and protamine by methyl methanesulfonate in the germ cells of male mice

The molecular dosimetry of methyl methanesulfonate (MMS) in the germ cells of male mice has been investigated. The mice were injected i.p. with 100 mg/kg of [3H]MMS and methylations per sperm head, per deoxynucleotide, and per unit of protamine were then determined over a 3-week period. The methylations per sperm head paralleled the dominant lethal frequency curve for MMS, reaching a maximum of between 22 and 26 million methylations per vas sperm head 8-11 days after treatment. Methylation of sperm DNA was greatest at 4 h (the earliest time point studied) after treatment, with 16.6 methylations/10(5) deoxynucleotides. DNA methylation gradually decreased during the subsequent 3-week period. The methylation of germ-cell DNA did not increase in the stages most sensitive to MMS (late spermatids leads to early spermatozoa) and was not correlated with the dominant lethal frequency curve for MMS. However, methylation of protamine did increase in the germ-cell stages most sensitive to MMS, and showed an excellent correlation with the incidence of dominant lethals produced by MMS in the different germ-cell stages. The pattern of alkylation produced by MMS in the developing germ-cell stages of the mouse is similar to that found for EMS. However, for equimolar exposures, MMS alkylates the germ cells 5-7 times more than does EMS. Hydrolyzed samples of protamine from [3H]MMS-exposed animals were subjected to thin-layer chromatography and amino acid analysis. Both procedures showed that most of the labeled material recovered from the hydrolysates co-chromatographed with authentic standards of S-methyl-L-cysteine. The amino acid analyses showed an average of approximately 80% of the labeled material eluting with S-methyl-L-cysteine. The mechanism of action of both MMS and EMS on the developing germ cells appears to be similar. The occurrence of S-methyl-L-cysteine as the major reaction product in sperm protamine after MMS exposure supports our initial model of how dominant lethals are induced in mouse germ cells by these chemicals: Alkylation of cysteine sulfhydryl groups contained in mouse-sperm protamine blocks normal disulfide-bond formation, preventing proper chromatin condensation in the sperm nucleus. Subsequent stresses produced in the chromatin structure eventually lead to chromosome breakage, with resultant dominant lethality.

Unscheduled DNA Synthesis in Mammalian Germ Cells—Its Potential Use in Mutagenicity Testing

The first evidence for unscheduled DNA synthesis (UDS) in mammalian somatic cells was provided by Rasmussen and Painter(1) when they demonstrated that radiation induced the uptake of labeled thymidine into the DNA of non-S-phase HeLa and Chinese hamster cells grown in culture. Later, Kofman-Alfaro and Chandley,(2) also using m vitro procedures, were able to demonstrate UDS in spermatogenic cells of mice that had been exposed to X-rays or UV. Similar findings were observed after in vitro UV exposure of rat germ cells(3) and of human germ cells.(4)

Unscheduled DNA synthesis in spermatogenic cells of mice treated in vivo with the indirect alkylating agents cyclophosphamide and mitomen.

Abstract Cyclophosphamide (CPA) and mitomen (DMO) are chemical mutagens that require metabolic activation to produce their biological effect. We have used an in vivo UDS assay in various meiotic and postmeiotic germ-cell stages of male mice to study DNA repair after treatment with these chemicals. EMS, a compound requiring no metabolic activation, was also used for comparative purposes. CPA and DMO induced UDS in meiotic through early-to-midspermatid stages, but no UDS was detected in late spermatids and mature sperm. While EMS produced a maximum UDS response in the germ cells immediately after treatment, CPA and DMO did not produce a maximum response until ∼0.5 to 1 h after injection. This delay is attributed to the time required for CPA and DMO to be enzymatically vonverted active alkylating metabolites. Unlike the results found with EMS, mutation frequencies (dominant lethals, translocations, specific-locus mutations) following CPA treatment are not noticeably reduced in germ-cell stages in which UDS occurred. In the case of DMO, mutations are induced only in mature spermatozoa, and these germ-cell stages represent only a fraction of those in which no UDS is detected. The results with CPA and DMO thus still leave unclear the relationship between DNA repair and the differential spermatogenic response of mice to genetic damage.

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.

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.