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Monomeric acrylamide was tested for its potential to induce aneuploidy in spermatocytes and bone marrow cells of mice. For this purpose, chromosomes from metaphase spreads were counted semi-automatically. In both test systems, cell proliferation was monitored, determining the meiotic index of spermatocytes and the average generation time of bone marrow cells after BrdU incorporation, respectively. No indications could be seen for different sensitivity of somatic and germinal cells towards acrylamide. With a dose of 120 mg/kg, the chemical caused cell cycle delay in both germ line and somatic cells. There was diverging response with respect to the balance of hypo- and hyperploidy. While the percentage of chromosome loss was significantly elevated in both test systems, acrylamide treatment did not increase the frequency of hyperploid cells. Interpreting these results on the basis of conventional test protocols, acrylamide should not be considered as an aneugen. The conservative approach, however, may be inadequate for the detection of aneugenic mechanisms different from non-disjunction.

The S9 fraction was prepared from rats i.p. injected with a single dose of 75 mg/kg acrylamide. The efficiency of the acrylamide induction procedure was verified on Salmonella mutagenicity testing (TA98 and TA100) using a number of known mutagens. These mutagens are aflatoxin B1, benzo[alpha]pyrene, 2-acetylaminofluorene and sodium azide. The activity of the mutagen is measured in terms of histidine revertant numbers which were found to be almost proportional to the concentration of acrylamide-induced S9. The maximum activation of the mutagens (except sodium azide) was recorded using around 50 microliters of S9 per plate. The efficiency of our prepared S9 fraction as a metabolic activator for some known mutagens is comparable to that of standard S9. On the basis of our results, acrylamide-induced S9 demonstrated considerable increase in the activation of the tested compounds. Accordingly, acrylamide could be used as an additional procedure for the induction of rat hepatic enzymes (S9).

The rodent spermatid micronucleus (MN) assay was used in conjunction with immunofluorescent techniques to distinguish kinetochores in MN following exposure of mice to X-radiation or acrylamide. After either treatment, modest increases in kinetochore-positive MN were observed. Spermatids which had been exposed during meiotic prophase to X-rays (400 cGy) had approximately 10-fold increases in MN compared to controls; up to 15% of the MN observed were kinetochore-positive. Following acrylamide treatment of meiotic prophase cells, there was a doubling of spermatid MN over baseline levels, approximately one-third of which were kinetochore-positive.

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.

Hamster embryos were treated with AF-2 in vivo (in the mother) by transplacental application. Their cells were then isolated, cultured in normal medium for 48 h, and then selected in media containing various concentrations of 8AG or 6TG. This treatment with AF-2 caused marked dose-dependent induction of 8AG- or 6TG-resistant mutants. Direct administration of AF-2 and MNNG to embryonic hamster cells also produced 8AG- and 6TG-resistant colonies. This new method is concluded to be very useful for detection of environmental mutagens and carcinogens when combined with the in vivo-in vitro combination assay of chemical carcinogenesis (transformation test).

The genetic activity of furapromidium (F30066), an antischistosomal drug, was studied in Salmonella typhimurium, Saccharomyces cerevisiae, Neurospora crassa and cultured Chinese hamster cells. The results show that F30066 induces gene mutations in S. typhimurium, N. crassa and Chinese hamster cells. This compound also causes gene conversions in S. cerevisiae.

Niridazole, one of several drugs presently known to be of value in the treatment of human schistosomiasis, was tested for its activity in inducing mitotic recombination in yeast. It was found that niridazole is genetically active when the treatment of yeast cells is performed in a rich medium (YPG-medium) under growing conditions, but not when treatment is carried out in a non-nutrient suspension (phosphate buffer). The data suggest that niridazole might be converted to an active compound by yeast metabolism. The results of the experiments with niridazole in the non-nutrient medium were compared with those of AF-2 and SQ18, 506, two agents which have been shown to be genetically active in the present assay system.

In a series of toxicity tests, male mice of three inbred strains were exposed to several doses of orally administered furylfuramide (AF-2). Subsequent to these test the effects of AF-2, as measured by induced dominant lethals, were tested in strain DBA/2J mice. AF-2 at the doses used in this study was relatively non-toxic to the strains of mice tested. No indication of AF-2 induced dominant lethality was observed.

Acrylamide induces chromatid exchanges and breaks with considerable frequency in spermatogonia of mice with long-term administration (3 weeks), though not, remarkably, with short-term administration (1--2 weeks. At 12 and 24 h after single injections with 50, 100 and 150 mg/kg acrylamide, evaluation of the cytogenetic effect is difficult in the spermatogonia because of an extreme reduction of mitotic cells. Aneuploid and polyploid cells increase with time after treatment in both marrow and spermatogonial cells, while the aberration frequency shows no increase in marrow after both oral-administration and injection. Evidently the spermatogonia are thus rather more sensitive to acrylamide than marrow cells. On the other hand, the SCE frequency is at the control level in treated subjects in marrow and spermatogonia. Acrylamide induces chain quadrivalents, ring quadrivalents, fragments and univalents which are particularly evident in primary spermatocytes in both oral administration and injection, though it is questionable whether these structural changes deal with spermatogonia, or otherwise with the S-phase primary spermatocytes. There is a possibility that the aberrant cells thus produced can develop into spermatozoa carrying a certain type of reciprocal translocation which leads to semi-sterile progeny. In relation to the above problem detailed investigations into this type of rearrangement in primary spermatocytes are needed.

Acrylamide and its 15 analogues have been tested for mutagenicity in 5 TA strains of Salmonella typhimurium. Acrylamide, N-tert-butylacrylamide, crotonamide, diacetone acrylamide, N,N-diethylacrylamide, N,N-dimethylacrylamide, N-hydroxymethylacrylamide, N-methylacrylamide, N-isobutoxymethylacrylamide, N-isopropylacrylamide, methacrylamide, N,N'-methylene-bis-acrylamide and N-tert-octylacrylamide appeared not to be mutagenic in the standard Ames assay both with and without Aroclor 1254-induced S9, and in both the plate incubation and liquid preincubation procedures. Three epoxide analogues, i.e., glycidamide, N,N-diglycidyl acrylamide and glycidyl methacrylamide showed mutagenicity in one or two strains both with and without the S9.

Monomeric acrylamide is an important industrial chemical primarily used in the production of polymers and copolymers. It is also used for producing grouts and soil stabilizers. Acrylamide's neurotoxic properties have been well documented. This review will focus on pertinent information concerning other, non-neurotoxic, effects observed after exposure to acrylamide, including: its genotoxic, carcinogenic, reproductive, and developmental effects. It will also cover its absorption, metabolism, and distribution. The data show that acrylamide is capable of inducing genotoxic, carcinogenic, developmental, and reproductive effects in tested organisms. Thus, acrylamide may pose more than a neurotoxic health hazard to exposed humans. Acrylamide is a small organic molecule with very high water solubility. These properties probably facilitate its rapid absorption and distribution throughout the body. After absorption, acrylamide is rapidly metabolized, primarily by glutathione conjugation, and the majority of applied material is excreted within 24 h. Preferential bioconcentration of acrylamide and/or its metabolites is not observed although it appears to persist in tests and skin. Acrylamide can bind to DNA, presumably via a Michael addition-type reaction, which has implications for its genotoxic and carcinogenic potential. The available evidence suggests that acrylamide does not produce detectable gene mutations, but that the major concern for its genotoxicity is its clastogenic activity. This clastogenic activity has been observed in germinal tissues which suggest the possible heritability of acrylamide-induced DNA alterations. Since there is 'sufficient evidence' of carcinogenicity in experimental animals as outlined under the U.S. EPA proposed guidelines for carcinogen risk assessment, acrylamide should be categorized as a 'B2' carcinogen and therefore be considered a 'probable human carcinogen.' The very limited human epidemiological data do not provide sufficient evidence to enable one to judge the actual carcinogenic risk to humans. Acrylamide is able to cross the placenta, reach significant concentrations in the conceptus and produce direct developmental and post-natal effects in rodent offspring. It appears that acrylamide may produce neurotoxic effects in neonates from exposures not overtly toxic to the mothers. Acrylamide has an adverse effect on reproduction as evidenced by dominant lethal effects, degeneration of testicular epithelial tissue, and sperm-head abnormalities.

The cytogenetic effects of 2(2-furyl)-3-(5-nitro-2-furyl)-acrylamide (AF-2), a food preservative used in Japan, on rat bone marrow cells in vivo were studied. The aberrant metaphase cells in the bone marrow increased and reached the peak level 6 h after intraperitoneal injection of 240 mg/kg body weight of AF-2 and returned to the normal level within 24 h. A dose-response relationship was obtained using 4-240 mg/kg of AF-2. Chromosome aberrations were also induced after oral administration of 30-240 mg/kg. The aberrations were mostly chromatid breaks and the distribution among and within chromosomes was similar to those induced by a carcinogen, 7, 12-dimethylbenz(a)anthracene (DMBA). The present results provide the first evidence of in vivo cytogenetic effects of AF-2 on mammalian cells and, together with the evidence of mutagenicity already proved in other organisms, warn of the possible genetic hazards to cells exposed to this compounds.

The genetic activity of the antimicrobial food additives AF-2 and H-193 has been investigated in Saccharomyces cerevisiae. The strains chosen for the present studies were D5 for the induction of mitotic recombinational events and XV185-14C for the induction of reversion of the mutants lys1-1, his1-7 and homs3-7. When three concentrations (25, 50 and 100 mug/ml) of AF-2 were used in the reversion system of strain XV185-14C, there was an increase in the frequency of hom+ and his+ revertants as a function of incubation time, while the lysine mutant exhibited a very low frequency of induced reversion. When AF-2 and H-193 were compared at the same concentration and exposure time, AF-2 exhibited a higher genetic activity in both systems than H-193. However, H-193 was genetically more active in inducing revertants than AF-2, when the comparison was made at the same survival level. Cells of both haploid and diploid strains were found to be more sensitive to inactivation by AF-2 than by H-193 It should be pointed out that the solubility of H-193 was lower (about 4 mug/ml saturation) than the solubility of AF-2 (120 mug/ml saturation). The haploid strain was more sensitive to both compounds than the diploid strain.

Trans-2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide (furylfuramide: FF or AF2) was tested for ability to induce 8-azaguanine (8AG) resistant mutations in cultured human diploid cells. FF had a relatively severe cytotoxic effect on the cells. From the concentration-survival curve, the D0 value for 2-h treatment with FF was estimated to be 11 mug/ml. When cells were treated with FF at various concentrations for 2 h, cultured in normal medium for 48 h, and then selected in medium containing 8AG at 30 mug/ml, the induced mutation frequency increased gradually with increase in concentration of FF. When cells were treated with FF at 10 mug/ml for 2 h, cultured in normal medium for various periods of mutation expression time, and selected with 8AG at 30 mug/ml, the highest induced mutation frequency was obtained with 48 h of mutation expression time. Microscopic examination of the numbers of cells in colonies indicated that the total number of cells increased by half during this mutation expression time of 48 h.

The Salmonella/microsome mutagenesis assay was used to determine the effect of cysteine (alpha-amino-beta-mercaptopropionic acid) on the mutagenic actions of several carcinogens: N-methyl-N'-nitro-N-nitrosoguanidine. N-acetoxy-2-acetylaminofluorene, N-hydroxy-2-acetylaminofluorene, 4-nitroquinoline-1-oxide, methyl methanesulfonate, 5-nitro-2-furaldehyde semicarbazone, 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide, aflatoxin B1 and the nitrosation products of methylurea and methylguanidine. Cysteine, at non-toxic concentrations, significantly decreased the frequency of reversion to histidine prototrophy when it was added to treatment mixtures. The extent of the inhibition of mutagenic action by cysteine depended on the carcinogen studied as well as the doses of cysteine and carcinogen employed. Cysteine (2.5--10 mM) completely inhibited the mutagenic actions of N-methyl-N'-nitro-N-nitrosoguanidine and methylguanidine nitrosation products while only partially preventing the mutagenic effects of the other carcinogens assayed. Inhibition of 5-nitro-2-furaldehyde semicarbazone-induced mutagenesis occurred only with higher cysteine concentrations (20--200 mM).