Ionizing radiation-induced instant pairing of heterochromatin of homologous chromosomes in human cells.

Using fluorescence in situ hybridization with human band-specific DNA probes we examined the effect of ionizing radiation on the intra-nuclear localization of the heterochromatic region 9q12-->q13 and the euchromatic region 8p11.2 of similar sized chromosomes 9 and 8 respectively in confluent (G1) primary human fibroblasts. Microscopic analysis of the interphase nuclei revealed colocalization of the homologous heterochromatic regions from chromosome 9 in a proportion of cells directly after exposure to 4 Gy X-rays. The percentage of cells with paired chromosomes 9 gradually decreased to control levels during a period of one hour. No significant changes in localization were observed for chromosome 8. Using 2-D image analysis, radial and inter-homologue distances were measured for both chromosome bands. In unexposed cells, a random distribution of the chromosomes over the interphase nucleus was found. Directly after irradiation, the average inter-homologue distance decreased for chromosome 9 without alterations in radial distribution. The percentage of cells with inter-homologue distance <3 micro m increased from 11% in control cells to 25% in irradiated cells. In contrast, irradiation did not result in significant changes in the inter-homologue distance for chromosome 8. Colocalization of the heterochromatic regions of homologous chromosomes 9 was not observed in cells irradiated on ice. This observation, together with the time dependency of the colocalization, suggests an underlying active cellular process. The biological relevance of the observed homologous pairing remains unclear. It might be related to a homology dependent repair process of ionizing radiation induced DNA damage that is specific for heterochromatin. However, also other more general cellular responses to radiation-induced stress or change in chromatin organization might be responsible for the observed pairing of heterochromatic regions.

Radioprotective effect of melatonin assessed by measuring chromosomal damage in mitotic and meiotic cells.

This study was taken to evaluate the radioprotective effects of melatonin. Male adult albino mice were treated (intraperitoneal, i.p.) with 10 mg/kg melatonin either 1 h before or 1/2 h after exposure to 1.5 Gy of gamma-irradiation. Control, melatonin, irradiated and melatonin plus irradiation groups were sacrificed 24 h following treatment. The incidence of micronuclei (MN) in bone marrow cells was determined in all groups. The results show that melatonin caused a significant reduction in micronuclei polychromatic erythrocytes (MNPCE) when animals were treated with melatonin before and not after exposure to radiation. Mitotic and meiotic metaphases were prepared from spermatogonial and primary spermatocytes, respectively. Examination and analysis of metaphases showed no mutagenic effect of melatonin on chromosomal aberration (CA) frequency in spermatogonial chromosomes. Administration of one single dose of melatonin to animals before irradiation lowered total CA from 46 to 32%. However, no significant effect was observed when melatonin was given after irradiation. Similarly, the frequency of CA in meiotic metaphases decreased from 43.5% in the irradiated group to 31.5% in the irradiated group treated with melatonin 1 h before irradiation, but no change was observed when melatonin was administered after irradiation. The data obtained in this study suggest that melatonin administration confers protection against damage inflicted by radiation when given prior to exposure to irradiation and not after, and support the contention that melatonin radioprotection is achieved by its ability as a scavenger for free radicals generated by ionizing radiation.

The mutagenic versus protective role of vitamin A on the induction of chromosomal aberration in human lymphocyte cultures.

The present study was carried out to evaluate the role of vitamin A (VA) on the induction of chromosomal aberrations (CA) in lymphocyte culture system and to investigate its modulating effect on chromosomal damage induced by gamma irradiation. Lymphocyte cultures from five healthy normal adult males were either treated with VA at a dose level of 2.0, 8.0 or 24.0 microg/ml or exposed to gamma-irradiation of 3.0 Gy, then followed immediately by a treatment with one of the above mentioned doses of VA. Non-treated cultures and cultures exposed to gamma-irradiation served as control for the two sets of experiments. Cultures were set up in duplicates and incubated for 48 h for assessment of CA. Treatment with VA alone increased CA demonstrating a dose-response effect. Addition of VA to gamma-irradiated cultures resulted in an inverse protective effect as the low dose of 2 microg/ml reduced the CA induced by radiation to about 1/3 rd whereas a dose of 8 microg/ml had a protective effect of 40% of the total damage and the large dose of 24 microg/ml had no or little effect. These results suggest that a proportion of the added VA may interfere with the radiation induced free radicals and other reactive metabolites which elevate CA. On the other hand, excessive amounts of VA increased toxicity and reduced effect on repair enzymes.

Chromosomal aberrations in chronic male alcoholics.

A group of 23 male heavy drinkers was studied cytogenetically, and the results were compared with those obtained from a control group of 50 non-drinkers. The data are based on karyological analysis of dividing lymphocytes in 72-hr blood cultures. Alcoholics revealed a significantly higher frequency of numerical and structural chromosomal aberrations. Chromosome rearrangements were observed only among alcoholics. The most frequent exchange type was dicentrics (0.02 per cell, constituting 65.1% of the total exchanges), followed by reciprocal translocations (0.005 per cell), and pericentric inversions (0.002 per cell). The significance of the presence of these types of chromosomal aberrations in the blood of alcoholics is discussed in terms of its prevalence and association with known syndromes and diseases. The possible effects and/or interactions of other factors such as duration of alcohol abuse, drugs, and smoking were also emphasized.

On the mutagenicity of methadone hydrochloride. Induced dominant lethal mutation and spermatocyte chromosomal aberrations in treated males.

The mutagenicity of methadone hydrochloride was tested in male mice using the dominant lethal mutation technique and the spermatocyte test of treated mice. Male mice of C3H inbred strain received one of the following doses, 1, 2, 4 or 6 mg/kg body weight once a day for 3 consecutive days. Another group of mice served as control and received saline instead. Treated males were then mated to virgin females at 3-day intervals for a period of 45 days. Pregnant females were dissected at mid-term and the corpora lutea and intrauterine contents were recorded. The spermatocytes of treated males were examined 45-50 d after treatments with methadone and abnormal pairing configurations were scored. The methadone treatment was found to increase the rate of preimplantation deaths consistently in all post-meiotic stages with all doses used. In addition, the higher doses, 4 and 6 mg, affected spermatogonia stages. Quantitatively, the dose-response relationship cannot be demonstrated though the spectrum of effect increased with higher doses as more spermatogenesis stages became more sensitive to the treatment. In many cases the frequency of live implants showed a positive correlation with preimplantation deaths in contrast with the frequency of early deaths which showed only sporadic variation. The mutation indices based on total embryonic death indicate that methadone hydrochloride affected several stages of germ-cell maturation namely, spermatozoa (M.I. 14-35), late spermatids (M.I. 15-48), early spermatids (M.I. 14-50), late spermatocytes (M.I. 15-43) and spermatogonial stages (M.I. 12-63). Chromosome analysis at diakinesis-metaphase 1 revealed significant increase in the frequency of sex chromosome and autosome univalents with different doses of methadone. The smallest dose applied was quite effective and the data represent direct dose-response relationship. Of the multivalent configuration, the most frequent type was chain quadrivalents. The frequencies of total translocations per cell were estimated as 0.1, 0.16 and 0.2 for the 4 applied doses illustrating a dose-response relationship for the doses: 1, 2 and 4 mg, whereas with the higher dose, 6 mg, an abrupt decrease was apparent (0.05). This study calls for concern regarding the possible genetic hazards this drug may impose upon human populations.

Role of the pituitary and the adrenals in mediating the effects of alcohol on testicular steroidogenesis in mice.

We have previously demonstrated that intragastric administration of alcohol (1.24 g/kg body wt) to adult male mice results in suppression of testosterone production. We now report that the decline in peripheral testosterone levels in alcohol-treated mice is not accompanied by changes in plasma levels of luteinizing hormone, follicle stimulating hormone or estradiol-17 beta, and that it is markedly attenuated in adrenalectomized or adrenalectomized-corticosterone treated males.

Studies in mice on the mutagenicity of two contraceptive drugs.

An experiment is described which tests for visible and invisible mutants in mice treated with four different doses each of the contraceptives Gynanovlar and Lyndiol. The results show that there is no reason to suppose that either substance has an appreciable mutagenic effect, expressed as an increase of antenatal and postnatal lethals or visibles. The substrain CBA/CagCam, used throughout, has an incidence of 0.27% of singly occurring abnormalities, mainly of the appendicular skeleton, which distinguishes it from the parent CBA strain and its axial variation described by Gruneberg (1963).

Suppression of testosterone production by ethyl alcohol. Possible mode of action.

Intragastric administration of ethyl alcohol (1.24 g/kg body weight) to adult male mice caused a drastic decrease in the concentration of testosterone (T) in peripheral plasma. The depression of plasma T levels was significant at 30, 60 and 90 minutes after alcohol administration, but by 120 min, the normal T levels were re-established. This transient decrease in peripheral T levels was probably due to a reduction in testicular T production, because at 1 hr after alcohol administration, the concentration of T in the testis was also significantly depressed. The ability of the testes of alcohol-treated mice to produce T in response to gonadotropic stimulation in vitro was not affected. Addition of 5, 10, 20 or 50 microliter of alcohol per ml of the medium used for the incubation of decapsulated testes had no significant effect on the accumulation of T, but similar doses of acetaldehyde caused a pronounced inhibition of T production. The decrease in plasma T levels observed after administration of ethyl alcohol in vivo may be related to a direct inhibition of testicular T production by acetaldehyde derived from the metabolism of alcohol.

Selection for high and low adrenal weight in mice.

Four strains of mice were used to found a heterogenous stock from which one line was selected for high adrenal weight/100 g body weight. By the eighth generation of selection, two stocks of mice were produced, one with adrenals whose absolute weight was greater than any of the parental strains. The realized heritability estimates were 75% for females and 67% for males during the period of selection, showing that a large component of variation in adrenal weight was genetic in origin. Asymmetry in response to selection was also noticed and attributed to possible variation in adrenal structure between sexes. Estimates of variances in each generation, particularly the eighth, did not show any decline which suggested that further responses to selection would be possible with further selection.

Effect of sexual maturation and androgens on prostaglandin levels in tissues of the male reproductive system in mice.

Prostaglandins E and F were measured in the testis, epididymis, vas deferens, and seminal vesicles of CD-1 mice from 2 to 8 weeks of age. The concentration of PGF was higher than that of PGE in all organs studied, except for the vas deferens. The concentration of prostaglandins (PGs) was age-dependent, showing a progressive decline from immaturity to adulthood. However, in the testis, there was an apparent transient increase in the concentration of PGs in the seminal vesicle changed very little between the ages of 5 and 8 weeks. The vas deferens had a significantly higher PG concentration than any of the organs studied, and a unique pattern of changes in the levels of PGE and PGF with age. In the vas deferens of two- and three-week-old mice, the concentration of PGF was higher than the concentration of PGE, but after 4 weeks of age PGE became somewhat more abundant than PGF. Treatment of immature mice with testosterone propionate (TP) produced significant changes in PG concentrations, resulting in PG levels resembling those of adult animals. The treatment also changed the ratio of PGE to PGF in the vas deferens (from 1:2 to 1:1). Hereditary dwarf mice had higher levels of PGs in the tissues of the male reproductive system than did their normal littermates. The treatment of dwarf mice with TP generally reduced the concentration of PGs in their reproductive system and resulted in a PG pattern more characteristic of normal adult males of the same strain. The data demonstrate pronounced changes in PG levels in the tissues of the male reporductive system of mice during sexual maturation. From the present study and from previous findings, it can be concluded that these changes can be accounted for by an increase in testicular testosterone secretion.

Effect of castration, testosterone treatment and hereditary sterility on prostaglandin concentration in the male reproductive system of mice.

The concentration of prostaglandins E and F in different parts of the male reproductive system of CD-1 and dwarf mice was measured by radioimmunoassay. In intact CD-1 mice, the vas deferens contained a significantly higher concentration of PGE and PGF than was found in the epididymis and in the seminal vesicles. All tissues studied had more PGE than PGF. Castration resulted in increased PG concentrations in both the epididymis and the seminal vesicles and decreased PG concentrations in the vas deferens. There was also a differential response of PGE and PGF in the epididymis of genetically sterile dwarf mice (dw/dw) were significantly higher than those observed in their normal littermates ((PLUS)). A reversed PGE/PGF ratio was found in the mates. The results indicate that testicular androgens affect the levels of PGE and PGF in the reproductive system of male mice. The physiological role of PGs in male reproductive functions has not been established, but there is a suggestion that PH have a role in controlling the transit of spermatozoa through the epididymis and vas deferens.

Prostaglandin levels in tissues of the male reproductive system in six strains of mice.

Prostaglandin E and F were measured in the testis, epididymis and vas deferens of six strains of inbred mice (A/J, AKR/J, C57BL/6J, DBA/2J, BALB/J and CE/J. The concentration of PGE and PGF was significantly higher in the vas deferens than in either the testis or the epididymis. Furthermore, the cauda epididymis contained a higher level of PGs than the caput portion. The relative preponderance of PGF to E varied between the tissues. The levels of PGs in the studies tissues indicated that they might be directly associated with the biological process of sperm maturation. The significant differences among strains of mice suggested that the observed variation might be, to a large extent, genetic.

Studies on the mutagenic effect of contraceptive drugs. I. Induction of dominant lethal mutations in female mice.

PIP: 30 adult virgin female mice (2 strains) received either high or low doses of Anovlar or Lyndiol oral contraceptives and were tested for induction of dominant lethal mutations. The pregnant mice were dissected on Day 14 of pregnancy and total implantations, early deaths, late deaths, and corpora lutea were counted in each pregnancy. A significant reduction in fertile mating (p .025) was found in 1 strain of those who received the high dose of Lyndiol (10 times that of the low dose, which is physiologically equivalent to the human dose). This dose also increased the number of dead implants in both strains which resulted in higher estimates of dominant lethal mutations. It is concluded that when Lyndiol and Anovlar were given at the physiological dose level to control ovulation in mice, the frequency of dominant lethal mutations was not increased above the control level.^ieng