Quantity of DNA in the organs of 19-day-old foetuses after AET, MEA, or 5-HT treatment of mice on the first day of gestation

On the first day of gestation, Porton mice were injected intraperitoneally with AET (2-aminoethylisothiouronium bromide hydrobromide), MEA (cysteamine hydrochloride,) or 5-HT (serotonin-creatinine sulphate), in a dose of 40 mg/kg of bodyweight. On the nineteenth day of pregnancy, the fresh weight of both heart and kidneys of foetuses, as well as DNA content in 25 mg of fresh tissue and in these whole organs were analysed. DNA was extracted from the foetal organs by means of Burton's method, which is based on the estimation of deoxiribose content in the colour reaction with diphenylamine. As compared to controls, in the remaining groups of mice lower fresh weight of both heart and kidneys of foetuses, greater DNA content in 25 mg of fresh tissue and smaller total amounts of DNA in the whole organs were found. Among the experimental groups of mice, statistically significant differences in the analysed values were observed between the group of animals treated with 5-HT and the remaining groups, with the exception of statistically non-significant difference in the DNA content of the whole kidneys between those injected with 5-HT and MEA.     

Embryonic survival in mice treated with AET, MEA or 5-HT on the first day of pregnancy

The embryotoxicity of AET, MEA, and 5-HT was investigated in Porton mice. Female mice on the first day of gestation were injected intraperitoneally with 2-amino-ethylisothiouronium bromide hydrobromide (AET), cysteamine hydrochloride (MEA) or serotonin-creatinine sulphate (5-HT) in a dose of 40 mg/kg body weight. Uterine contents were examined on the nineteenth day of pregnancy. As compared with controls, in mice treated with AET, MEA or 5-HT, a smaller number of live fetuses and a greater number of non-implanted embryos, resorptions, and dead fetuses were found. Not all females which were injected with these compounds had live fetuses. Among the compounds, MEA appeared to be more toxic than AET and 5-HT.     

Effects of AET and MEA treatment of mice on the first day of pregnancy

Porton female mice were injected intraperitoneally with 2-aminoethylisothiouronium bromide hydrobromide (AET) or cysteamine hydrochloride (MEA) in a dose of 40 mg/kg of body weight on the first day of pregnancy. On the last, nineteenth, day of gestation, taking into consideration females in whose uterus live fetuses were observed, the increase in their body weight throughout pregnancy, the number of fetuses in the uterus, the body weight of fetuses, and placental weight were found smaller in mice treated with AET or MEA, than in control ones. Among the injected compounds, AET appeared to be less toxic than MEA.     

Induction of micronucleated erythrocytes by MEA,AET, WR-2721 and X-rays

The induction of micronucleated polychromatic erythrocytes (MNPCEs) was assessed in the bone marrow of adult male Swiss mice treated with MEA (cysteamine HCl), AET (2-aminoethylisothiouronium Br.HBr), or WR-2721 (S-2-(3-aminopropylamino)ethyl phosphorothioic acid), at a dose of 200 mg/kg body weight, and/or exposed to 6 Gy X-rays. MEA, AET, or WR-2721 was given alone or 15 min prior to X-ray exposure, and the frequency of MNPCEs was determined 24 h after the aminothiol treatment and X-irradiation of mice. A genotoxic effect was shown for MEA, AET, WR-2721, and X-rays, as well as a protective effect of the aminothiols against X-ray-induced genotoxicity in the mouse erythropoietic system. The aminothiol drugs given alone, without subsequent X-irradiation, elevated the frequency of MNPCEs, and WR-2721 appeared to be less toxic than AET and MEA. After exposure of mice to X-rays, the number of MNPCEs was distinctly increased. MEA, AET, or WR-2721 administration prior to X-irradiation resulted in a reduction of the X-ray-induced elevation of the frequency of micronuclei, but a stronger radioprotective effect was obtained following WR-2721 and AET treatment than after MEA application. So, the genotoxic and radioprotective effect of the aminothiols was dependent on the compound applied.     

Acid phosphatase and arylsulphatase activities in testes after AET or MEA treatment of adult mice.

Temporal changes of acid phosphatase (E.C. 3.1.3.2) and arylsulphatase (E.C. 3.1.6.1) activities in testes of adult Swiss mice after AET (2-amino-ethylisothiouronium Br. HBr) or MEA (cysteamine HCl) treatment, were studied. The animals were injected intraperitoneally with the S-containing substances in a single dose of 400 mg/kg body weight. The enzyme activities in crude organ homogenates were assessed every four hours during a 24-hour period. Administration of the aminothiol agents to mouse organism caused greater changes in the acid phosphatase activity than in the arylsulphatase activity, and the two chemical compounds AET and MEA given, influenced the enzyme activities in testes in a different way. Treatment of mice with AET resulted in a decrease of the acid phosphatase activity related to 1 g of fresh tissue at 16.00 and the whole organ weight at 24.00 and 16.00 as well as in a decrease of the arylsulphatase activity expressed per the whole weight of testes at 08.00. After MEA injection, the acid phosphatase activity related to 1 mg of protein, 1 g of fresh tissue and the whole organ weight was decreased at 20.00(1), and the enzyme activity expresse per 1 mg of protein and 1 g of fresh tissue was increased at 24.00, but the arylsulphatase activity related to both 1 mg of protein at 08.00, 12.00 and to the whole weight of testes at 08.00, was reduced.     

Liver and muscle glycogen contents and blood glucose concentration after AET or MEA treatment of adult male mice.

A statistically significant decrease was found in the glycogen content expressed in mg per 1 g of both liver in groups of mice killed in the 60th and 90th minute, and skeletal muscles in those killed in the 30th, 60th, and 90th minute after MEA injection, as well as a statistically significantly reduced blood glucose concentration in a group of mice killed in the 30th minute after AET treatment.     

Quantity of glycogen in the liver of 19-day-old foetuses after AET, 5-HT, MEA, or GSH treatment of pregnant mice on the first day of gestation

Swiss mice were treated intraperitoneally with AET, 5-HT, MEA, or GSH, in a dose of 80 mg/kg of body weight, on the first day of gestation. On the 19th day of pregnancy, the fresh weight of liver of the foetuses, as well as glycogen content in 1 g of fresh tissue and in the whole organ were analysed. The determination of glycogen content in the foetal liver were made according to the anthrone method. As compared with controls, in the remaining groups of mice a lower fresh weight of foetal liver less glycogen per g of fresh tissue and a smaller total amount of glycogen in the whole organ were found. Among the compounds, AET appeared to be more toxic than 5-HT, MEA, and GSH.     

Temporary changes of protein level in liver after AET or MEA treatment prior to 60Co gamma-irradiation of male mice

The adult male Swiss mice were either whole-body gamma-irradiated with a single dose of 10 Gy from 60Co source, always at 19.00 or, 15 minutes before irradiation injected intraperitoneally with AET (2-aminoethylisothiouronium Br. HBr), or MEA (cysteamine HCl), in a dose of 400 mg/kg body weight. The measurements of the protein level in crude homogenates of liver were done in four-hour internals during a 24-hour period, starting at 20.00. The protein concentration in liver was calculated per 1 g of fresh tissue and the whole organ weight. The body and liver weight was also studied. There were no fluctuations in the liver weight and concentration of protein in the control and irradiated only mice. Temporary changes in the liver weight and level of protein expressed in mg per 1 g of fresh tissue and the whole organ weight could be found in the group of males treated with AET, and daily changes in the liver weight and concentration of protein related to mg per 1 g of fresh tissue, in the group of male mice injected with MEA prior to irradiation, could be recorded. Differences in the liver weight at 20.00, 24.00 and 04.00, as well as in the protein level expressed in mg per 1 g fresh tissue at 04.00, 12.00, 16.00, and the whole liver weight at 24.00, 04.00, and 16.00, of between the particular groups of mice, were observed. There were no temporary changes in the body weight in any of the groups and there were no differences in this value between the groups of mice.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of radioprotectors on the cAMP and cGMP systems

Summary The sulphur-containing radioprotectors mercaptoethylamine (MEA), aminoethylisothiourea (AET), 2-aminothiazoline, 4-oxo-2-aminothiazoline, and S-S-3-oxapentane-1,5-diisothiourea, and the radioprotective biogenic amines serotonin, histamine, and dopamine, caused the elevation of cAMP content and intensified the rate of cAMP-dependent protein phosphorylation in tissues of animals following intraperitoneal injection at radioprotective doses. Biogenic amines stimulated the adenylate cyclase activity in membrane preparations from liver, spleen, and small-intestine mucosa; sulphur-containing radioprotectors caused no such effects. None of the radioprotectors affected cAMP and cGMP phosphodiesterases in vitro. AET and MEA inhibited guanylate cyclase in vitro, whereas serotonin and dopamine stimulated the enzyme. A biphasic change in the level of cGMP was observed in tissues after the administration of MEA and AET (more than 2-fold fall by 1–3 min after the administration of drug and 1.4-fold rise after 15–20 min); serotonin and dopamine caused a slow rise in the cGMP level; the cAMP/cGMP ratio in liver showed biphasic changes in level during the 20 min following injection of serotonin.The data obtained support the conclusion that the action of radioprotectors on cellular metabolism in animals may be mediated by the cAMP system. The reciprocal regulation of radioresistance by cAMP and cGMP is unlikely to exist.     

Effects of AET and MEA on the lysosomal hydrolase activities in the mouse organs.

The adult male Swiss mice were injected intraperitoneally with AET (2-aminoethylisothiouronium Br.HBr) or MEA (cysteamine HCl), in a toxic dose of 400 mg/kg body weight. The acid phosphatase (E.C. 3.1.3.2) and arylsulphatase (E. C. 3.1.6.1) activities in crude homogenates of liver and kidneys were assessed every fourth hour throughout a 24-h period. Different patterns of temporal changes in the acid phosphatase and arylsulphatase activities in liver and kidneys expressed in nkat per 1 mg of protein, 1 g of fresh tissue and per the whole organ weight, were found. The extent and timing of the alterations in the activity of each of the lysosomal hydrolases were dependent on the particular organ chosen and aminothiol compound given.     

Polysaccharides induce radioprotection of murine hemopoietic stem cells and increase the LD50/30 days

Intravenous administration of 60 mg/kg of a polysaccharide (MNR, MNZ, GLP/BO4, GLP/BO5) significantly decreases the mortality of mice exposed to a single dose of X rays. The dose reduction factors (DRF) obtained for MNZ, MNR, GLP/BO4, and GLP/BO5 given intraperitoneally 15 min before exposure were 2.16, 1.93, 1.80, and 1.94, respectively. The DRF was not increased when MNZ or GLP/BO4 were combined with injection of AET before X-ray exposure. The LD50 for the CFUs exposed in vivo in mice was 1.13 Gy for the treated mice and 0.75 Gy for the nontreated mice. This corresponds to a DRF of 1.6. The DRF calculated from the slope is 1.27.     

Chemical radioprotection of hemopoietic colony-forming cells: comparative effect of AET, anoxia, and urethan

The bone marrow colony-forming unit (CFU) technique of Till and McCulloch was employed to test the radioprotective effect of AET, anoxia, urethan on marrow cells irradiated in vivo. For AET and anoxia, a dose-reduction factor of 1.9 to 2.1 was found. Since the marrow cells were assayed for CFU content immediately after irradiation of the donor, the observed effect can be interpreted as a "true" radiation dose reduction. By contrast, urethan injection did not increase the survival of marrow CFU assayed immediately after whole-body x-irradiation. However, both urethan and AET afforded radioprotection of endogenous CFU content of spleen and bone marrow, but not of endogenous spleen colony count. It is concluded that the mechanism of radioprotection by urethan is fundamentally different from that of AET or anoxia.     

Differential radioprotection of tissues in C3H/J mice by a combination of 5-hydroxy L-tryptophan with 2-aminoethylisothiuronium bromide hydrobromide.

Differential radioprotection between normal tissues and carcinoma was observed in C3H/J mice treated with a combination of 5-hydroxy L-tryptophan (5-HTP, 100 mg/kg) and 2-aminoethylisothiuronium bromide hydrobromide (AET, 20 mg/kg). Protection to normal tissues was judged by LD50(30) and by radiation induced damage to bone marrow(BM) using clonogenic ability of blood forming stem cells (10 day CFUs) as the criteria. Pretreatment with 5-HTP + AET combination 30 min before whole body gamma radiation (WBGR) enhanced the recoveries of the number of blood forming stem cells in BM of irradiated mice after 0, 7th and 10th day of irradiation. LD50(30) for C3H/J mice was 7.3 Gy and the dose modifying factor (DMF) of 5-HTP + AET combination was 1.76. On the contrary, pretreatment with this combination did not protect the mammary carcinoma transplanted in C3H/J mice, when exposed to 80 Gy soft X-rays.     

Value of chemical mixture in multiple supralethal X-irradiation of mice

A chemical mixture containing 25 Amoles of MEA, 7 pmoles of AET, and 2 micromoles of 5-HT was found to be of significant value in protecting mice against repeated exposures of 800 R, 1100 R, or 1400 R given at intervals of 28 days. Dose-reduction factors of 2.17, 2.18, 1.95, 2.14, and 1.58 were obtained for the first five exposures. Following the first exposure there was no chemical mortality. The beneficial value of this mixture, however, was limited by the incidence of chemical toxicity which was more prevalent in mice with higher cumulative doses of radiation.     

Inhibitory effects of technical grade methoxychlor on development of neonatal male mouse reproductive organs

Effects of technical grade methoxychlor (MX), an estrogenic insecticide, and 17 beta-estradiol (E2) were examined on serum testosterone (T) concentrations and growth and histology of neonatal male mouse reproductive organs. Male NIH/Swiss mice received i.p. injections daily from birth to Day 9 with one of the following: 10 micrograms E2 or 0.1 or 1.0 mg MX or sesame oil vehicle. The mice were killed on Day 10. MX or did not affect body weights or mortality. Serum T concentrations in control mice were 0.29 +/- 0.03 ng/ml; T concentrations in males treated with MX or E2 were reduced. The 1.0-mg dose of both MX and E2 significantly decreased DNA contents of the seminal vesicles (SV), bulbourethral glands (BUG), and ventral prostate (VP) compared to controls. In the same animals, DNA contents of testes, epididymides, and efferent ductules were not decreased. The lower dose of MX (0.1 mg) decreased DNA content of only BUG and SV. DNA content of the ductus deferens was not affected by any treatment. E2 and both doses of MX also decreased epithelial morphogenesis in the SV and BUG, and inhibited the onset of mucin production in BUG epithelium and smooth muscle differentiation in the ductus deferens. In summary, our results indicate that technical grade MX, at doses as low as 0.1 mg/day, and E2 inhibit neonatal male reproductive tract development and decrease serum T concentrations.     

Differential recovery of intestine, bone marrow, and thymus of rats with solid tumors following 5-fluorouracil administration.

Time relationships for recovery of several host organs from toxic effects of 5-fluorouracil were determined in ACI rats bearing Morris hepatoma 3924A. A single injection of 150 mg/kg body weight 5-fluorouracil (the LD10) resulted in loss of 90% of the tibial bone marrow, 60% of the intestinal mucosa, and 90% of the thymus as measured by total DNA content of the organs. Organ DNA contents following 150 mg/kg of the drug were minimal on day 3 for intestine and on day 5 for marrow and thymus. A return to pretreatment or higher levels of DNA was observed by day 4 for intestine, day 11 for tibial marrow, and day 19 for thymus. Incorporation of 3H-deoxyuridine into host organ DNA after 150 mg/kg 5-fluorouracil was inhibited 36 hrs for intestine, 3 days for thymus, and 5 days for tibial bone marrow. Inhibition of 3H-deoxyuridine incorporation into DNA was similar for 50, 100, and 150 mg/kg doses both in tumor and in host organs, but recovery of 3H-deoxyuridine incorporation and DNA content of host organs began later with the higher doses of 5-fluorouracil. Maximal incorporation of 3H-deoxyuridine into DNA was observed on day 4 for intestine, day 8 for marrow, and day 9 for thymus after treatment with 150 mg/kg 5-fluorouracil. Animal lethality following the second of two 150 mg/kg injections of 5-fluorouracil was related to the extent of recovery of intestinal mucosa and bone marrow at the time of the second injection. Survival decreased to 0% for normal rats when the interval between injections was 3-4 days, improved at 5 days and was 100% when the interval was 10-11 days.     

Role of selenium on antioxidant capacity in methomyl-treated mice

Methomyl carbamate is a pesticide widely used in the control of insects. The present work aims at studying the effect of selenium on the antioxidant system of methomyl-treated mice. Swiss albino mice were intraperitoneally administered a single dose of methomyl (7 mg/Kg body weight). Mice of another group were injected with sodium selenite (5 pmole/Kg b.wt.) 7 days before methomyl intoxication. After 24 hours, methomyl exposure resulted in significant increase in lactic dehydrogenase activity (LDH). The antioxidant capacity of hepatic cells in terms of the activities of superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), glutathione-S-transferase (GST) and glutathione (GSH) content was diminished. It appears that methomyl exerts its toxic effect via peroxidative damage to hepatic, renal and splenic cell membranes. Also, methomyl induced DNA damage in these organs as detected by alkaline filter elution technique. The distribution of methomyl in different organs of mice was detected by HPLC. Selenium administration prior to methomyl injection produced pronounced protective action against methomyl effects. It is observed that selenium enhances the endogenous antioxidant capacity of the cells by increasing the activities of SOD, CAT, GR and GST as well as increasing GSH content. The activity of LDH was decreased in blood and the damage of DNA was suppressed comparable to controls. In conclusion, the adverse effects of methomyl in mice could be ameliorated by selenium.     

Chemical protection against the long-term effects in mice exposed to supralethal doses of X rays

Our results demonstrate that mixtures of radioprotectors increase the degree of protection against the short and the long term effects compared with that obtained with each substance given separately. The most potent mixtures of radioprotectors (AET, MEA, Cyst, GSH, 5-HT) yield for the long term survival a dose reduction factor of 2.1. Pulmonary lesions are most often the cause of death in protected mice irradiated with 13.5 Gy or more. At the time of death signs of sclerosis and atrophy in several tissues are associated with these lung lesions in most mice and increase with dose and time after exposure. The tissues most affected are the kidney, the alimentary tract, the liver and the lymphoid tissues.     

Effect of safranal, a constituent of Crocus sativus (saffron), on methyl methanesulfonate (MMS)-induced DNA damage in mouse organs: an alkaline single-cell gel electrophoresis (comet) assay

The influence of safranal, a constituent of Crocus sativus L. stigmas, on methyl methanesulfonate (MMS)-induced DNA damage was examined using alkaline single-cell gel electrophoresis (SCGE), or comet, assay in multiple organs of mice (liver, lung, kidney, and spleen). NMRI mice were divided into five groups, each of which contained five mice. The animals in different groups were received the following chemicals: physiological saline (10 mL/kg, ip), safranal (363.75 mg/kg, ip), MMS (120 mg/kg, ip), safranal (72.75 mg/kg, ip) 45 min prior to MMS administration, and safranal (363.75 mg/kg, ip) 45 min prior to MMS administration. Mice were sacrificed about 3 h after the administration of direct mutagen MMS, safranal, or saline, and the alkaline comet assay was used to evaluate the influence of safranal on DNA damage in different mouse organs. Increase in DNA migration was varied between 9.08 times (for spleen) and 22.12 times (for liver) in nuclei of different organs of MMS-treated mice, as compared with those of saline-treated animals (p < 0.001). In control groups, no significant difference was found in the DNA migration between safranal- and saline-pretreated mice. The MMS-induced DNA migration in safranal-pretreated mice (363.75 mg/kg) was reduced between 4.54-fold (kidney) and 7.31-fold (liver) as compared with those of MMS-treated animals alone (p < 0.001). This suppression of DNA damage by safranal was found to be depended on the dose, and pretreatment with safranal (72.75 mg/kg) only reduced DNA damage by 25.29%, 21.58%, 31.32%, and 25.88% in liver, lung, kidney, and spleen, respectively (p < 0.001 as compared with saline-treated group). The results of the present study showed that safranal clearly repressed the genotoxic potency of MMS, as measured by the comet assay, in different mouse organs, but the mechanism of this protection needs to be more investigated using different in vitro system assays and different experimental designs.     

Protective effect of Crocus sativus stigma extract and crocin (trans-crocin 4) on methyl methanesulfonate-induced DNA damage in mice organs

This study was designed to examine the effect of aqueous extract of Crocus sativus stigmas (CSE) and crocin (trans-crocin 4) on methyl methanesulfonate (MMS)-induced DNA damage in multiple mice organs using the comet assay. Adult male NMRI mice in different groups were treated with either physiological saline (10 mL/Kg, intraperitoneal [ip]), CSE (80 mg/Kg, ip), crocin (400 mg/Kg, ip), MMS (120 mg/Kg, ip), and CSE (5, 20, and 80 mg/Kg, ip) 45 min prior to MMS administration or crocin (50, 200, and 400 mg/Kg, ip) 45 min prior to MMS administration. Mice were sacrificed about 3 h after each different treatment, and the alkaline comet assay was used to evaluate the effect of these compounds on DNA damage in different mice organs. The percent of DNA in the comet tail (% tail DNA) was measured. A significant increase in the % tail DNA was seen in nuclei of different organs of MMS-treated mice. In control groups, no significant difference was found in the % tail DNA between CSE- or crocin-pretreated and saline-pretreated mice. The MMS-induced DNA damage in CSE-pretreated mice (80 mg/Kg) was decreased between 2.67-fold (kidney) and 4.48-fold (lung) compared to those of MMS-treated animals alone (p < 0.001). This suppression of DNA damage by CSE was found to be depended on the dose, which pretreatment with CSE (5 mg/Kg) only reduced DNA damage by 6.97%, 6.57%, 7.27%, and 9.90% in liver, lung, kidney, and spleen, respectively (p > 0.05 as compared with MMS-treated group). Crocin also significantly decreased DNA damage by MMS (between 4.69-fold for liver and 6.55-fold for spleen, 400 mg/Kg), in a dose-dependent manner. These data indicate that there is a genoprotective property in CSE and crocin, as revealed by the comet assay, in vivo.