Dominant Lethal Mutation Assay Research Articles

Reproductive toxicity assessment of Olusosun municipal landfill leachate in Mus musculus using abnormal sperm morphology and dominant lethal mutation assays.

Mixture of xenobiotics in Olusosun landfill leachates (OSL) induced somatic and germ cell genotoxicity in eukaryotic cells. However, whether the DNA-damaged germ cells can increase reproductive failure, embryotoxicity and/or teratogenicity during fertilization remained unclear. The study herein investigated reproductive toxicity and embryotoxic effects of OSL in mice using abnormal sperm morphology and dominant lethal mutation assays. Six mice per group were exposed to 0.5 mL of 5, 10, 25, and 50% OSL (v/v, leachate/distilled water) for 5 consecutive days. Similar treatment was giving to distilled water and cyclophosphamide (20 mg/kg body weight), as negative and positive control groups, respectively. At 35 day post-treatment, the mice were sacrificed and examined for cauda epididymal abnormal sperm morphology, testicular weight change and histopathological lesions. Male mice were mated to untreated nulliparous females (1 treated male per 2 untreated females) until vaginal plugs were observed. Pregnant females were sacrificed on 14th day post-mating and uterine content examined for number of implants per female, resorptions, and live and dead fetuses. OSL significantly increased teratozoospermia by 31.02% with amorphous sperm head having the highest frequency and sperm with two heads, the least. Histology of the testes revealed congestion of the interstitial blood vessels, spermatid retention and disorganization of the germinal epithelium from the basal compartment. OSL insignificantly increased absolute and relative testes weights but reduced body weight of exposed mice. There was insignificant induction of dominant lethal mutation, although low fertility was observed in the treated mice. The analyzed physico-chemical parameters and heavy metals/metalloids in OSL were higher than permissible limits. The metals, physico-chemical parameters and other unanalyzed substances in OSL induced abnormal sperm morphology, altered testes pathology, but did not significantly induce dominant lethal mutation in mice. Constituents of OSL are toxic to the male reproductive organs in mice and suggests harmful impact on public health. Indiscriminate disposal of solid wastes in the environment should be prevented.

English

Dominant lethal mutation assay was carried out on rats after being treated with graded doses of ethanol extract of the seeds of Mucuna urens. Male albino rats (Wistar strain) were caged in three groups labeled, groups II, III and IV and treated with three different dosages of the ethanol extract of the seeds of M. urens; 70, 140 and 210 mg/kg body weight (BW), respectively, for 14 days. The positive control animals (group I), were treated with distilled water for the entire period. At the end of the feeding period of two weeks, they were co-habited with virgin female albino rats at a ratio of 1:1 for 3 days. 14 days after mating, the females were sacrificed for the dominant lethal mutation assay. The results of the dominant lethal mutation assay showed that only female rats in group II had implants on the uterine horn, of all the treated groups. The rats in groups III and IV did not have any implants at all. Biological evaluations (pre-implantation losses) carried out showed 0, 76, 100 and 100% lethal mutations in groups I, II, III and IV, respectively. The statistical evaluations obtained showed 8.6b ± 0.47, 6.6a ± 0.94, 0 ± 0.0 and 0 ± 0.0 in groups I, II, III and IV, respectively. Photographs of the corpora lutea were obtained using a digital camera (DCR-HC48E, KODAK). The results obtained can be attributed to the induction of dominant lethal mutations in spermatocytes and early spermatids in the male Albino rats, showing the mutagenic effect of the seeds of the plant M. urens and its potential as a male contraceptive. Key words: Mucuna urens seeds, dominant lethal mutations assay, mutagenesis, male Albino rats.

Chronic ethanol consumption in mice does not induce DNA damage in somatic or germ cells, evaluated by the bone marrow micronucleous assay and the dominant lethal mutation assay

Although alcohol is known to be a carcinogen for humans, ethanol-genotoxicity studies are incomplete. Ethanol seems not to be a bacterial mutagen, but the results are conflicting in rodent assays. We investigate the genotoxicity in the bone marrow micronucleus (MN) test and in the dominant lethal mutation (DLM) assay using two long-term ethanol exposure protocols. In the MN test, mice consumed three doses (5, 10 and 15% v/v) for 32 weeks. MN induction was compared to two control groups of 5- and 38-week-old mice (the ages of the treated mice when the treatment was initiated and when they were killed, respectively). For the three groups treated with ethanol there was no significant increase in MN induction as compared to the first control group, but observed MN frequencies were significantly lower than in the 38-week-old control group. This suggests a protective effect against genotoxic damage caused by aging, probably due to ethanol action as a hydroxyl radical scavenger. In the DLM assay, male mice drank ethanol at 15% or 30% (v/v) for 20 weeks. In both groups the number of dead implants was similar to the control, but there was a significant reduction in total implants, indicating a pre-implantation loss.

Genotoxicity of crotonaldehyde in the bone marrow and germ cells of laboratory mice

Genotoxicity of crotonaldehyde was evaluated by employing bone marrow and spermatocyte chromosomal aberration and dominant lethal mutation assays in Swiss albino mice. For bone marrow chromosomal aberration assay, animals were treated with 0.2 ml olive oil containing 8, 16 and 32 μl kg b.w. of crotonaldehyde for 6, 12 and 24 h through single intraperitoneal injection. Treatment induced dose-dependent and statistically significant decrease in mitotic index and increase in chromosome aberrations per cell (CAs/cell excluding gaps and stickiness and pulverizations) and percent aberrant metaphase (excluding gaps) in the bone marrow cells at 6, 12 and 24 h after treatment. For spermatocyte chromosomal aberration assay, male mice were treated with 8, 16 and 32 μl/kg b.w. of crotonaldehyde for 24 h through single intraperitoneal injection. The percentage of the induced chromosome aberrations in diakinesis-metaphase-I cells showed dose-dependent increase. For dominant lethal mutation assay, adult male mice were injected intraperitoneally with 0.2 ml olive oil containing crotonaldehyde at the rate of 8, 16 and 32 μl/kg b.w. for 5 consecutive days. The negative control animals received 0.2 ml olive oil as above. In the dominant lethal mutation assay after the last dose, the treated males were allowed to mate with untreated virgin females. The mating continued for 5 consecutive weeks. At pregnancy days 14–16, the females were killed and uterine contents were examined for live and dead implants. Treatment of mice resulted in statistically significant decrease in the fertility indices and total number of implants per female. Statistically significant decrease in the number of live implants per female and increase in the number of dead implants per female were recorded in females mated with males during 8–14 and 15–21 and 22–28 days post-treatment mating. Crotonaldehyde treatment of males induced appreciably higher frequencies of dominant lethal mutation during 8–14, 15–21 and 22–28 days post-treatment mating periods. Percent dominant lethal mutation increased concomitantly with the dose. Dominant lethality was maximum in females mated with males treated with 5 × 32 μl/kg b.w. during the 15–21 days post-treatment mating. The overall result suggests a positive dose–response relationship between treatment and induction of chromosomal aberrations in the somatic and germ cells and dominant lethal mutation in the germ cells.

Genotoxicity of lomefloxacin—an antibacterial drug in somatic and germ cells of Swiss albino mice in vivo

The in vivo genotoxicity of lomefloxacin, a diflourinated antibacterial drug, was evaluated by employing mouse in vivo chromosomal aberration test in bone marrow cells and dominant lethal mutation assay in germ cells. Statistically significant reduction in mitotic index, increase in chromosomal aberrations (CAs)/cell and percent abnormal metaphase was observed only at the highest dose (160 mg/kg b.w.) of the drug. In the dominant lethal mutation assay, a statistically significant decrease in the number of implants/female, compared to vehicle control, was noticed only in the females mated with males treated with 32 mg/kg b.w. during the third week of mating, while statistically significant reduction in live implants/female was noticed at both the doses during the second and third weeks of mating. Nevertheless, no significant change in the number of dead implants/female was observed after lomefloxacin treatment. These results seems to indicate that lomefloxacin is a weak clastogen in the bone marrow cells and non-mutagenic in the germ cells of mouse in vivo.

Mutagenic profiles of carbazole in the male germ cells of Swiss albino mice

Mutagenic effect of carbazole was evaluated by employing dominant lethal mutation and sperm head abnormality assays in male Swiss albino mice. For the dominant lethal mutation assay, adult male mice were treated for five consecutive days either with 30 or 60 mg/kg body weight (b.w.) of carbazole by single intraperitoneal (i.p.) injection. For the sperm head abnormality assay mice were treated with 50, 100, 150, 200 and 300 mg/kg b.w as a single i.p. injection. Treatment of adult male mice with carbazole resulted in induction of dominant lethal mutation and abnormal sperm heads. The results show that carbazole is mutagenic in male germ cells of mice.

Germ cell mutagenicity of phthalic acid in mice

Mutagenicity of phthalic acid was evaluated by employing dominant lethal mutation and sperm head abnormality assays in male Swiss albino mice. For the dominant lethal mutation assay, adult male mice received a single intraperitoneal (i.p.) injection of either 40 mg or 80 mg/kg b.w. of phthalic acid for 5 consecutive days. For the sperm head abnormality assay, the mice were treated with 50, 100, 150, 200 and 300 mg/kg b.w. as a single i.p. injection. Treatment of adult male mice with phthalic acid resulted in induction of dominant lethal mutations and abnormal sperm heads. The results obtained indicate that phthalic acid is a germ cell mutagen.

Sperm head abnormalities and dominant lethal effects of formaldehyde in albino rats

The effects of formaldehyde exposure have been investigated in rats by two short-term in vivo mutagenicity tests (sperm head abnormality and dominant lethal mutation assays). Five daily interperitonial injections of formaldehyde resulted in a statistically significant increase in induction of sperm head abnormalities at 0.125 to 0.500 mg/kg. The frequency of dominant lethal mutations in female rats sired by males exposed to formaldehyde was significantly higher than the control group. There was also a reduction of fertile matings in females mated 1–7 days after treatment of males with formaldehyde. The genetic and environmental health implications of the results are discussed.

Fate of unfertilized ova in male rodent dominant lethal assays: Extension of the studies by Kratochvilova

Kratochvilova has described a technique whereby ova can be recovered from mated mice and their stage of division determined. This is of value to determine if reduced total implantations in a male dominant lethal (DL) germ cell mutation assay are due to pre-implantation loss of embryos, a presumed mutagenic event, or to chemically induced male infertility. Kratochvilova was not specific about the fate of unfertilized ova, but it was implied that they undergo a process of fragmentation that might be confused with the regular cleavage of fertilized ova. It became important for us to draw a firm distinction between ova fragmentation and regular ova cleavage in the rat. We therfore repeated the ova analyses of female mice mated with males exposed to iso-propyl methanesulphonate (iPMS), as described by Kratochvilova. Following that calibration study the technique was extended to the rat via ova cleavage analysis in mated female rats, coupled to a study of the normal decay of ova in virgin rats. Unfertilized ova are shown to undergo irregular fragmentations that can be clearly distinguished from normal cell division. It is concluded that the individual or combined incidences of single celled ova and fragmented ova (dependent on the cleavage stage of the concurrent control embryos) can provide a measure of male infertility as it relates to reduced implantations in DL assays. This ability to regard two morphological classifications of unfertilized ova as providing evidence for male infertility will simplify the conduct of ova analyses in both the mouse and the rat.

Incidence of spontaneous post-implantation deaths in CFT-Swiss mice: its suitability to assess dominant lethal mutations.

The primary objective of the study was to determine the incidence of spontaneous post-implantation deaths (DI) in our randomly-bred closed colony of CFT-Swiss mice. These data are required mainly to ascertain the suitability of the strain for their use in dominant lethal (DL) mutation assays, a well validated in vivo mammalian germ cell assay. To achieve the first goal, randomly drawn healthy adult males were sequentially mated to adult virgin females (1:2 ratio) for a period of 8 weeks. Male-based analysis of the 3 major variables viz., implantations, live embryos, and dead implants (DI) were carried out. Absence of outliers and low variation in spontaneous DI, as well as the other variables associated with DL assay were observed in all matings. Secondly, the degree of induction of DI were studied following administration (i.p.) of an acute dose of ethyl methane sulphonate (EMS). EMS induced nearly a 5-fold increase in the DI during the first 2 weeks and a 3-fold during the third week suggesting its DL-type effect only in specific post-meiotic stages of spermatogenesis as reported earlier.

Activity of iPMS and nPMS in mouse bone marrow micronucleus assays: comparison with mouse dominant lethal assay data

isoPropyl methanesulphonate (iPMS) and its n-propyl analogue (nPMS) are shown to be active in mouse bone marrow micronucleus assays using male CBA, male and female ( C3H El × 102 El )F1 and male and female Muta ™Mouse mice. iPMS was significantly more active than nPMS. No significant strain or gender differences were observed. These findings reflect the differences reported earlier for these two chemicals in mouse dominant lethal mutation assays. The earlier described dominant lethal assay data are represented schematically and discussed.

Cytogenetic, genetic, and embryotoxicity studies with dimethyl 2,2,2-trichloro-1-(2,2,2-trichloro-1-hydroxyethoxy)-ethylphosphonate, a hypothetical impurity in technical grade trichlorfon

The hemiacetal (CH 3O) 2P(O)CH(OCHOHCCl 3)CCl 3, a hypothetical contaminant in technical preparations of the organophosphorus pesticide trichlorfon, was tested for cytogenetic, mutagenic, and embryotoxic activity after ip administration to mice of different strains. A single dose of 81 mg/kg (0.2 mmol/kg) caused a significant enhancement in the percentage of chromosome aberrations in bone marrow cells of CFLP mice; a similar effect was induced by an equimolar single dose of chemically pure trichlorfon (51.5 mg/kg). At the same dosage level, the hemiacetal proved to be ineffective in the micronucleus test on fetal blood of DBA and AB Jena/Halle mice. In the dominant lethal mutation assay, a single dose of 81 mg/kg hemiacetal to males resulted in a slight increase in the fetal mortality of DBA mice, whereas AB Jena/Halle mice did not respond under these conditions. Four consecutive doses of 81 mg/kg hemiacetal to pregnant AB Jena/Halle mice at Days 2, 3, 4, and 5 of gestation caused only a very weak embryotoxic effect comparable to that of trichlorfon at equimolar dosage. On the basis of these results the hemiacetal tested may not be considered to represent a potential risk factor in technical grade trichlorfon.

Mutagenic efficiency of organophosphorus insecticides used in combined treatments.

Male mice (Q strain) received two consecutive injections of organophosphorus insecticides: a phosphonate (trichlorfon) was combined to a thiophosphate (methylparathion) or a dithiophosphate (malathion or methylazinphos) in order to evaluate the interactions at the genetic and cytogenetic levels. No increase in chromosome damage was observed in bone marrow cells, spermatogonia, and primary spermatocytes. In a dominant lethal mutation assay, the frequency of postimplantation lethality was not significantly increased over the control level. The percentage of preimplantation losses was enhanced, probably due to a toxic effect on male germ cells.

Cytogenetic and genetic effects of subchronic treatments with organophosphorus insecticides.

Male mice (Q strain) received 5 days a week for 7 weeks drinking water containing dichlorvos (2 ppm), dimethoate (0.6 ppm), malathion (8 ppm), methylparathion (0.15 ppm), or trichlorfon (0.5 ppm). At the end of the treatment, no chromosome damage was observed in bone marrow cells, spermatogonia, and primary spermatocytes. Dominant lethal mutation assays were performed to investigate the pre- and postimplantation foetal lethality. Only negative results were obtained.

Absence of genetic and cytogenetic effects in mice treated by the organophosphorus insecticide parathion, its methyl analogue, and paraoxon

Male mice (Q strain) received a single i.p. injection of 3 organophosphorus compounds: ethylparathion (10 mg/kg), its methyl analogue (methylparathion, 10 mg/kg), or its phosphate derivative (ethylparaoxon, 0.3 mg/kg). The numver of chromosome aberrations observed in bone marrow cells and spermatogonia, and the frequency of pre- and postimplantation foetal lethality obtained in a dominant lethal mutation assay, did not conclusively prove that the tested compounds produced a mutagenic effect.

Evaluation of the mutagenic potential of four commercial mixtures of insecticides

Male mice (Q strain) were given a single ip injection at the maximum tolerated dose of one of four commercial mixtures of insecticides: Luxan Tue-Taons (150 g dimethoate and 150 g fenitrothion/litre), Metadipterex (210 g trichlorfon and 270 g methyldemeton/litre), Dynafos (155 g malathion, 60 g dichlorvos and 75 g carbaryl/litre) and Phosan Plus (95 g dimethoate, 100 g malathion and 100 g methoxychlor/litre). At the maximum tolerated doses, Luxan Tue-Taons (60 mg/kg), Metadipterex (15 mg/kg), Dynafos (50 mg/kg) and Phosan Plus (100 mg/kg) did not induce chromosome aberrations in bone-marrow cells, spermatogonia or primary spermatocytes of the mice. No evidence of potential genetic effects was obtained in a dominant lethal mutation assay.

Genetic and cytogenetic effects induced in the mouse by an organophosphorus insecticide: Malathion

Male mice (Q strain) received an ip injection of malathion (300 mg/kg). The percentage of chromosome aberrations was not increased in both bone marrow cells and spermatogonia. In a dominant lethal mutation assay, the frequency of pre- and postimplantation fetal lethality was not significantly enhanced over the control level.

Genetic and cytogenetic effects of Fenitrothion.

Male mice (Q strain) were injected intraperitoneally with a high dose (i.e., 1 g/kg) of the organophosphorus insecticide Fenitrothion. No increase in the percentage of chromosome aberrations was observed in bone marrow cells and spermatogonia. A dominant lethal mutation assay did not show any enhancement of fetal mortality before or after implantation.