P.S. Chauhan

Effect of vanillin on methylene blue plus light-induced single-strand breaks in plasmid pBR322 DNA.

The ability of vanillin (4-hydroxy-3-methoxybenzaldehyde), a naturally occurring food flavouring agent, in inhibiting photosensitization-induced single-strand breaks (ssbs) in plasmid pBR322 DNA has been examined in an in vitro system, independent of DNA repair/replication processes. Photosensitization of DNA with methylene blue, visible light and oxygen, induced ssbs resulting in the production of open circular form (OC form) in a concentration-dependent manner. The yield of OC form induced by photosensitization was increased several-fold by deuteration of the buffer and was found to be inhibited by sodium azide, a scavenger of singlet oxygen (1O(2)). Vanillin, per se, did not induce but inhibited photosensitization-induced ssbs in plasmid DNA, at millimolar concentrations. The inhibitory effect of vanillin was both concentration- and time-dependent. On a molar basis, vanillin was, however, less effective than trolox, a water-soluble analogue of alpha-tocopherol. Photosensitization by methylene blue system generates singlet oxygen, as one of the major components of ROS. Therefore, interaction of singlet oxygen with vanillin was investigated. The rate constant of vanillin with 1O(2) was estimated to be 5.93x10(7)M(-1)s(-1) and that of sodium azide as 2. 7x10(8)M(-1)s(-1). The present investigations show that vanillin can protect against photosensitization-induced ssbs in the plasmid pBR322 DNA, and this effect may partly be due to its ability to scavenge 1O(2).

The effect of hycanthone and maleic hydrazide on the frequency of micronuclei in the bone-marrow erythrocytes of mice

Male Swiss mice were assigned to 6 groups of either 3 or 4 animals each. 3 groups were given hycanthone methanesulfonate intraperitoneally, at 40, 80 or 120 mg/kg, respectively; the dose was repeated after an interval of about 24 h. At the same time 2 groups received maleic hydrazide at 100 or 200 mg/kg, and the remaining group was given dimethyl sulfoxide which was used as a solvent for both drugs. 6 h after the second injection, the mice were killed and bonemarrow preparations were made. Hycanthone induced a significant increase in the frequency of micronuclei in the polychromatic erythrocytes and suppressed the P/N ratio significantly. However, there was no dose-response relationship. Maleic hydrazide, on the other hand, failed to influence the incidence of micronuclei or the ratio of poly- to normo-chromatic erythrocytes.

Dominant lethal mutations in male mice fed γ-irradiated diet

Three groups of Swiss male mice were fed a stock ration or an unirradiated or irradiated (2·5 Mrad) test diet for 8 wk. After the feeding period, the males were mated with groups of untreated female mice for 4 consecutive weeks. The females were autopsied at mid-term pregnancy for evaluation of dominant lethal mutations. Numbers of dead implantations, including deciduomas and dead embryos, showed no significant differences among the different groups, thus producing no evidence of any induced post-implantation lethality in mice fed on irradiated diet. Similarly, there was no indication of pre-implantation lethality, since implantation rates remained comparable among different groups. Consumption of irradiated diet did not affect the fertility of mice. Total pre- and post-implantation loss, as indicated by the numbers of live implantations remained comparable among all the groups of mice.

Failure of ethanol to induce dominant lethal mutations in wistar male rats

2 groups of Wistar male rats (6-7 weeks old) were given ethanol, 15% in drinking water, for 5 days. The level of ethanol was gradually raised to 20 and 30% resp. Both groups received ethanol between 15 and 20% or 15 and 30% continuously during 35 days. Another group was given 30% ethanol for a period of 4 days before mating started. In addition to a control group, which received no treatment, a positive control group of rats exposed to 200 R X-rays, was used. After the treatments, individual males were paired with virgin Wistar females (10-12 weeks) at weekly intervals, and 8 sequential pairings were undertaken. Females were examined for uterine contents and corpora lutea, 10-11 days after their separation from the males. The females mated with irradiated males showed a high incidence of dead implantations and reduction of live implantations. No significant differences in the number of dead, live and total implantations at pre- and/or post-implantation levels were observed among the control and the ethanolic groups, showing that ethanol did not induce any dominant lethal mutations in these Wistar male rats. In the light of studies on alcoholics and other recent data, a need to investigate, independently, the potential mutagenic effects of ethanol and alcoholic beverages is discussed.

Quinacrine dihydrochloride, the non-surgical female sterilant induces dicentrics, rings, and marker chromosomes in human peripheral blood lymphocytes treated in vitro: a preliminary report

During the last decade, quinacrine dihydrochloride (QDH) has been promoted for clinical trials as a much needed non-surgical female sterilant, largely in the Third World. Recently, however, these human trials have come under severe criticism due to lack of adequate evidence of biological safety of QDH, particularly of its genotoxicity in mammalian systems. In the present study, the cytogenetic analysis of QDH-treated human lymphocytes, grown as whole blood cultures in vitro, surprisingly showed a wide range of chromosomal aberrations. At a concentration of 3.0 and 6.0 microg/ml in culture, QDH was cytotoxic, as shown by the very few analyzable metaphases that could be observed. G(0) lymphocytes, treated with 0. 6 microg/ml QDH, exhibited chromosome aberrations including dicentrics, ring configurations, translocations, inversions, and marker chromosomes. Near haploid, polyploid, and endoreduplicated cells were also observed. All the rings appeared to be formed as a result of telomere fusion/association. Twenty percent of the dicentrics observed also indicated telomere fusion/association in the D and G groups of chromosomes. Overall, a frequent involvement of chromosomes 1, 2, and 3 in both unstable and stable chromosome rearrangements was also observed. Exposure of 72-h cultures to 0.45 microg/ml QDH at 69 h resulted in an accumulation of C-metaphases, suggesting that probably QDH behaves as a mitotic spindle inhibitor. The G(2) lymphocytes from two donors exposed to 0.6, 1.5 or 3.0 microg/ml of QDH showed no increase in chromatid aberrations in two donors. However, QDH at 0.6 microg/ml increased the frequency of micronucleated binucleate cells. No increase in sister chromatid exchanges was observed at this concentration. Though preliminary, these observations demonstrate the chromosome damaging ability of QDH in human lymphocytes treated in vitro. Surprisingly, like ionizing radiation, QDH acted by an S-phase-independent mechanism, unlike most of the chemical mutagens. These results warrant detailed investigations on the cytogenetic effects of QDH in vitro, as well as among women exposed to this agent during clinical trials for non-surgical sterilization. The interesting cytogenetic profile of QDH deserves to be pursued and the underlying mechanisms, in particular, the DNA topoisomerase II inhibitory effect, if any, needs to be elucidated.

Studies on the effect of ethanol on dominant lethal mutations in Swiss, C57BL6 and CBA mice.

Swiss, C57BL6 and CBA males were given 0.1 ml of 40% ethanol per mouse per day for three consecutive days, intraperitoneally. These males were mated with untreated virgin Swiss females employing a 4-day mating schedule and three consecutive matings were carried out. In another study, C57BL6 males were given an ascending gradient of 5% to 40% ethanol in drinking water for a total period of 11 weeks. These males were mated with C57BL6 females for 2 weeks. Females were dissected at mid-term pregnancy for the examination of uterine contents including total, live and dead implants. All the investigations comprised at least two or three independent experiments which were evaluated independently as well as after pooling the data. Swiss, C57BL6 and CBA males given 0.1 ml of 40% ethanol, intraperitoneally, gave no evidence of any significant increase in post-implantation lethality in the postmeiotic phase of spermatogenesis attributable to ethanol treatment. A moderate but significant reduction in mean total implants indicating pre-implantation losses was seen in Swiss but not in CBA mice. Prolonged feeding of ethanol up to 40% in drinking water failed to provide any evidence of dominant lethal mutations in C57BL6 males at the pre-implantation level and the post-implantation lethals were also not significantly higher than in controls. In Swiss mice, however, the mutagenic index based on both pre- and post-implantation lethality was consistently positive.

Evaluation of freshly irradiated wheat for dominant lethal mutations in Wistar rats.

Three independent, serially performed experiments involving acute and chronic feeding of freshly irradiated wheat (75 krad, gamma-irradiation) were carried out in Wistar rats. In the first experiment groups of 10 males were given wheat for 1 week; irradiated wheat was consumed by the animals within 24 h or irradiation. In the other two experiments feeding of males was continued for 6 (10 males per group) and 12 (13 males per group) weeks, respectively, and the irradiated wheat was fed within 7 days of irradiation. At the end of each treatment period each male was paired with 3 females for 7 days and sequentially at weekly intervals for 5 or 8 weeks. Females were killed and examined for live and dead implantations and corpora lutea. There were no differences between groups with regard to fertility nor was there any inter-group differences as regards pre- and post-implantation losses whether the rats were fed irradiated or non-irradiated wheat. This suggested that even feeding of freshly irradiated wheat does not induce any dominant lethal mutations in rats.