Effects of long-term mildronate treatment on cardiac and liver functions in rats.

Mildronate is a cardioprotective drug that improves cardiac function during ischaemia and functions by lowering l-carnitine concentration in body tissues and modulating myocardial energy metabolism. The aim of the present study was to characterise cardiovascular function and liver condition after long-term mildronate treatment in rats. In addition, changes in the plasma lipid profile, along with changes in the concentration of mildronate, l-carnitine and gamma-butyrobetaine were monitored in the rat tissues. Wistar rats were perorally treated daily with a mildronate dose of either 100, 200 or 400 mg/kg for 4, 8 or 12 weeks. The l-carnitine-lowering effect of mildronate was dose-dependent. However, the carnitine levels reached a plateau after about four weeks of treatment. During the additional weeks of treatment, the carnitine levels were not considerably changed. The obtained results provide evidence that even a high dose of mildronate does not alter cardiovascular parameters and the function of isolated rat hearts. Furthermore, the histological evaluation of liver tissue cryosections and measurement of biochemical markers of hepatic toxicity showed that all the measured values were within the normal reference range. Our results provide evidence that long-term mildronate administration induces significant changes in carnitine homeostasis, but it is not associated with cardiac impairment or disturbances in liver function.

[Anti-atherosclerotic action of mildronate in experiment]. / Antiateroskleroticheskoe deístvie mildronata v éksperimente.

Mildronate is a new antiischemic drug which inhibits biosynthesis of carnitine from butyrobetaine. We studied antiatherosclerotic and antiinflammatory effects of mildronate in guinea-pigs, rats and rabbits. We found a hypolipidemic action of this substance in rats with triton WR-1339 hyperlipidemia. A protective antiatherosclerotic effect of mildronate was observed in rabbits kept on the atherogenic diet for 3 months. Antiinflammatory efficiency of mildronate in rats was demonstrated after injection of bradykinin, carragenine or implantation of a small cotton-wool pad.

Evaluation of 2-(methylaminosulfonyl)-1-(arylsulfonyl)-1-methylhydrazines as anticancer agents.

Seven new 2-(methylaminosulfonyl)-1-(arylsulfonyl)-1-methylhydrazines were prepared. The anticancer activity of these compounds was assessed in murine Ehrlich ascites carcinoma (EAC) by in vivo screening. Moderate in vivo activity in EAC was exhibited by three compounds. All of them were screened in vitro against a battery of human tumor cell lines at the National Cancer Institute (NCI), USA. One of them, compound 3a has displayed highly significant specificity in the renal tumor cell line RXF 393. These three compounds were also assessed for in vitro anti-HIV activity at the NCI, however, they have not reached the criteria of significant activity. The alkylating activity of the compounds was determined by measuring the absorbance of the alkylated product of 4-(4-nitrobenzyl)pyridine. It has been found that they are capable of acting as chemical alkylating agents.

Mechanism of site-specific DNA damage induced by methylhydrazines in the presence of copper(II) or manganese(III).

DNA damage induced by methylhydrazines (monomethylhydrazine, 1,1-dimethylhydrazine, and 1,2-dimethylhydrazine) in the presence of metal ions was investigated by a DNA sequencing technique. 1,2-Dimethylhydrazine plus Mn(III) caused DNA cleavage at every nucleotide without marked site specificity. ESR-spin-trapping experiments showed that the hydroxyl free radical (.OH) is generated during the Mn(III)-catalyzed autoxidation of 1,2-dimethylhydrazine. DNA damage and .OH generation were inhibited by .OH scavengers and superoxide dismutase, but not by catalase. The results suggest that 1,2-dimethylhydrazine plus Mn(III) generates .OH, not via H2O2, and that .OH causes DNA damage. In the presence of Cu(II), DNA cleavage was caused by the three methylhydrazines frequently at thymine residues, especially of the GTC sequence. The order of Cu(II)-mediated DNA damage (1,2-dimethylhydrazine greater than monomethylhydrazine approximately 1,1-dimethylhydrazine) was not correlated with the order of methyl free radical (.CH3) generation during Cu(II)-catalyzed autoxidation (monomethylhydrazine greater than 1,1-dimethylhydrazine much greater than 1,2-dimethylhydrazine). Catalase and bathocuproine, a Cu(I)-specific chelating agent, inhibited DNA damage while catalase did not inhibit the .CH3 generation. The order of DNA damage was correlated with the order of ratio of H2O2 production to O2 consumption observed during Cu(II)-catalyzed autoxidation of methylhydrazines. These results suggest that the Cu(I)-peroxide complex rather than the .CH3 plays a more important role in methylhydrazine plus Cu(II)-induced DNA damage.

Enzyme-altered foci in colons of carcinogen-treated rats.

The distal colon and rectum from male F344 rats treated with 15 mg/kg 1,2-dimethylhydrazine.2HCl (DMH) for 20 weeks were analyzed for focal areas of enzyme alteration. Tissues were embedded in methacrylate at 4 degrees C and cut in 2- to 4-micron serial sections. In DMH-treated rats, 8.8 +/- 2.4 foci/cm2 of examined mucosa were observed at 20 weeks and 7.7 +/- 1.1 foci/cm2 at 31 to 52 weeks, compared with 1.2 +/- 0.6 foci/cm2 in control rats (P = 0.01). The number of foci at 31 to 52 weeks compared with 20 weeks did not change significantly, but the area of altered rectal mucosa increased from 0.22 +/- 0.2% at 20 weeks to 1.47 +/- 0.6% at 31 to 52 weeks (P = 0.051). Most foci had decreased N-acetyl-beta-D-glucosaminidase, alpha-naphthyl butyrate esterase, and mucin in epithelial cells and increased gamma-glutamyl transpeptidase in the stroma. Morphologically, the foci varied from normal to overtly dysplastic. Grossly, tumors were identified in 5 of 20 DMH-treated rats killed at 31 to 52 weeks but not in 12 DMH-treated rats killed at 20 weeks or 30 control rats killed at 20 to 52 weeks. These data suggest but do not establish that enzyme-altered foci are putative preneoplastic lesions in the colon.

Effect of 1,1-dimethylhydrazine on lymphoproliferation and interleukin 2 immunoregulatory function.

The studies reported here suggest that the immunomodulatory effects of 1,1-dimethylhydrazine (UDMH) are associated, in part, with interference with interleukin 2 (IL-2) regulatory action. Concanavalin A (Con A)-stimulated DNA synthesis in murine splenocytes was inhibited from 18.6 to 44.1% at sub-toxic concentrations of UDMH (10 to 50 micrograms/ml) and IL-2-dependent DNA synthesis in CTLL-20 cells was inhibited from 11.3 to 41.58% at sub-toxic concentrations of UDMH (10 to 50 micrograms/ml). In addition, UDMH suppressed phorbol myristic acetate (PMA)-stimulated IL-2 production in EL-4 cells by up to 30% and slightly suppressed IL-2 production by Con A-stimulated murine splenocytes. In all cases, inhibition was evident at sub-toxic UDMH concentrations and was demonstrated to be independent of inactivation of IL-2 or interference with IL-2 absorption. It is suggested that UDMH has the potential to modify immune function through interference with IL-2 production and especially the lymphoproliferative response to IL-2.

Toxicity of N-nitrosodimethylamine, N-nitrosomethylbenzylamine, and 1,2-dimethylhydrazine in isolated rat hepatocytes.

N-Nitrosodimethylamine and 1,2-dimethylhydrazine were shown to injure lethally primary monolayer cultures of rat hepatocytes only after incubation periods in excess of 24 hr. The toxic action of these agents, therefore, mimics the time dependency of their hepatoxicity in vivo. The viability of hepatocytes treated with N-nitrosomethylbenzylamine was not different from controls at times up to 54 hr following treatment, a result which is also consistent with the inability of this compound to produce hepatotoxicity in vivo.

[The induction of cancer of the large intestine in Macaca fascicularis monkeys with 1,2-dimethylhydrazine]. / Induktsiia raka tolstoi kishki u obez'ian Macaca fascicularis 1,2-dimetilgidrazinom.

Continuous subcutaneous administration of 16 mg/kg body weight 1,2-dimethylhydrazine three times a month (total dose--1080-3696 mg) to Macaca fascicularis monkeys induced cancer invariably confined to the colon within 34-47 weeks. Biologic, clinical, histologic features and natural course of the tumor proved similar to those of its human counterpart.

Tumor induction in mice administered neonatally with 1,2-dimethylhydrazine.

1,2-Dimethylhydrazine was given subcutaneously to neonatal ICR mice, and animals were observed for one year. The tumors of lung and liver, malignant lymphoma, and other tumors were induced. In the mice given DMH 30 mg/kg of body weight, two mice developed colonic tumor, and the incidence of colonic tumor was low.

Dose- and time-response of colon carcinogenesis in Fischer-344 rats after a single dose of 1,2-dimethylhydrazine.

The purpose of this experiment was to examine the development of colon tumors after the injection of a single dose of 1,2-dimethylhydrazine (DMH) in rats. Male Fischer-344 rats were injected with 0, 10, 30, 100 or 200 mg/kg of DMH dihydrochloride. No tumors were seen after 3 or 5 months at any dose, but were seen after 7 or 9 months. At 9 months, tumors were induced in a dose-dependent manner, with 64.7% of rats receiving the 200 mg/kg dose developing tumors. This study shows that a high incidence of colon tumors can be induced by a single dose of DMH after a 9-month latency period.

Inositol and inositol hexaphosphate suppress cell proliferation and tumor formation in CD-1 mice.

In previous studies, we have shown that inositol hexaphosphate (InsP6), a constituent of cereal diet, inhibited azoxymethane-induced experimental large intestinal cancer (LIC) in Fischer 344 rats. We now report a similar antineoplastic action of InsP6 in CD-1 mice injected with 1,2-dimethylhydrazine (DMH). We had hypothesized that InsP6 may bring about this effect by undergoing dephosphorylation to lower phosphorylated forms; the ready availability of Ins, to react with phosphates, may increase the total amount of the lower phosphorylated Ins and potentiate the action of InsP6. LIC induced by DMH (15 mg/kg/week x 13) in mice given a mixture of 1% InsP6 + 1% Ins show a significant reduction (P less than 0.005) in LIC prevalence over InsP6 treatment. Surprisingly, Ins, an in vitro growth promoting agent also caused a significant (P less than 0.001) suppression of LIC. InsP6 +/- Ins also showed a concomitant reduction in the mitotic rate in the non-neoplastic epithelium. Body weight data did not suggest any overt toxic effect of long-term administration of InsP6, Ins or InsP6 + Ins. Since InsP6 is antineoplastic in two species of experimental animals, it should, in combination with Ins, be considered in our strategies for prevention of large intestinal cancer.

Inhibition of 1,2-dimethylhydrazine-induced nuclear damage in rat colonic epithelium by chlorophyllin.

Chlorophyllin (CHL) was tested for its effect on 1,2-dimethylhydrazine (DMH)-induced nuclear aberrations in rat colonic epithelium. Rats were given water containing CHL (1.5 mM) and food ad libitum. After 5 wks on this dietary regimen, the rats were given a single intraperitoneal injection of DMH (20 mg DMH base/kg body weight). The rats were sacrificed 18 hours later and their colons were removed. Ten consecutive crypts from the proximal and distal portion of each specimen were scored for nuclear aberrations and the karyorrhectic index (KI) was determined. Rats receiving CHL + DMH had significantly fewer nuclear aberrations (lower KI) in the colonic epithelium than rats given DMH alone. This implies that CHL, a known antimutagen, may have anticarcinogenic properties as well.

Chloroform inhibition of 1,2-dimethylhydrazine-induced gastrointestinal tract tumors in the Fisher 344 rat.

The effect of chloroform (CHCl3), administered at 0, 900, and 1800 mg/liter in the drinking water, on the carcinogenic potency of 1,2-dimethylhydrazine (DMH) was investigated. Groups of 40 male Fisher 344 rats were given one of the three drinking water solutions for 39 weeks following the subcutaneous injection of 200 mg/kg DMH, a known gastrointestinal (GI) tract carcinogen in this animal strain. When tumors from the GI tract were pooled there was a highly significant (p less than 0.001) decrease in total number of tumors per group with increasing concentration of drinking water CHCl3. In the control group (0 mg/liter CHCl3), 14/39 (36%) of the animals developed tumors of the GI tract, including the duodenum, jejunum, stomach, cecum, and colon. In contrast, the incidence of tumors in the two groups of rats given CHCl3 in the drinking water was significantly lower (p less than 0.001; 900 mg/liter CHCl3, 12.8%; 1800 mg/liter CHCl3, 12.5%). A similar relationship was obtained when colon tumors were analyzed independently (p = 0.01). The incidence of total colon tumors obtained in the control group of this study (10/39, 26%) agrees well with the previous study by B.S. Reddy, K. Watanabe, and J.H. Weisburger (1977, Cancer Res. 37, 4156-4159) conducted in the same rat strain (7/30, 23%). These results demonstrate that CHCl3 in the drinking water inhibits carcinogenesis in the rat GI tract.

1,2-Dimethylhydrazine-induced alterations in N1-acetylspermidine levels in rat distal colonic mucosa: effects of 2-difluoromethylornithine.

Recently, our laboratory has demonstrated that N1-acetylspermidine levels were increased in the distal colonic mucosa of rats administered 1,2-dimethylhydrazine for 15 and 26 weeks. In order to further explore the possible role of this acetylated polyamine in the malignant transformation process induced by this carcinogen, groups of rats were subcutaneously injected weekly with dimethylhydrazine (20 mg/kg body wt.) or diluent for 5, 10, 15 and 26 weeks +/- 1% 2-difluoromethylornithine in the drinking water. The latter agent, an irreversible inhibitor of ornithine decarboxylase, has previously been shown to inhibit colonic tumor formation in this experimental model. At each of these time periods, rats from each group were killed, their proximal and distal colonic mucosa harvested and examined, and compared with respect to polyamine levels, including N1-acetylspermidine, as well as the activities of ornithine decarboxylase, S-adenosylmethionine decarboxylase, spermidine N1-acetyltransferase and polyamine oxidase. The results of these experiments demonstrated that: (1) N1-acetylspermidine levels in the proximal colonic segment of all animals were similar at each time point; (2) N1-acetylspermidine levels were also similar in the distal colons of all animals at 5 and 10 weeks. At 15 weeks, however, the level of N1-acetylspermidine was increased in the dimethylhydrazine-treated distal colonic segment secondary to increases in the activity of spermidine N1-acetyltransferase; and (3) at 26 weeks, the level of this acetylated polyamine remained higher in dimethylhydrazine-treated distal 'uninvolved' colonic mucosa and was markedly elevated in colonic tumors; (4) co-administration of difluoromethylornithine decreased the elevated levels of N1-acetylspermidine to control values in the distal colons of animals treated with carcinogen for 15 and 26 weeks; and (5) difluoromethylornithine markedly reduced the number of tumors induced by dimethylhydrazine in the distal but not proximal colonic mucosa at 26 weeks.

Suppression of a a carcinogen (1,2-dimethylhydrazine dihydrochloride)-induced increase in mitotic activity in the colonic crypts of rats by addition of dietary cellulose.

Serial injections of the colon carcinogen, 1,2-dimethylhydrazine (DMH), have been reported to increase the proliferative activity in the colonic crypts preceding development of tumors. Can addition of purified cellulose to a fiber-free AIN-76 rat diet be used to suppress this increase in proliferative activity? To answer this question rats were divided into two groups, and one group was given eight weekly injections of the DMH base at 9.5 mg/kg of body weight. Throughout this period and for 2 additional wk the rats were isocalorically fed a defined nutritionally complete diet both with and without different dietary levels of cellulose (0, 5, and 15%). The rats were given injections of colchicine 3 h prior to sacrifice to arrest and to collect dividing cells at metaphase. Analysis of variance was performed on various morphometric parameters obtained from histological sections of midaxial crypts from the descending colon. Our results confirm that DMH induced a significant increase in the mitotic activity as measured by the number of metaphase figures per crypt. The presence of dietary cellulose did cause a significant suppression of the DMH-induced increase in the crypt mitotic activity.

Genetic factors controlling inheritance of susceptibility to 1,2-dimethylhydrazine.

Reciprocal crosses were made between AKR/J, a 1,2-dimethylhydrazine (DMH)-resistant mouse strain, and SWR/J, a sensitive strain. The F1 hybrids were tested with DMH and methylazoxymethanol (MAM), two colon carcinogens. Either DMH (20 mg/kg body weight) or MAM (35 mg/kg body weight), a metabolic derivative of DMH, was injected weekly for 10 weeks. In each group of 35 mice, 10 were injected with tritiated thymidine (25 microCi) 1 week after the sixth injection of DMH and MAM for the evaluation of proliferative characteristics and the number of foci of dysplasia occurring in 325 microns of distal colonic mucosa. At 27 weeks after the first injection of the carcinogen, the colons of remaining mice were opened longitudinally and the number of tumors enumerated. Compared with DMH-treated mice, the number of foci of dysplasia per mouse, the percentage of tumor-bearing mice, the number of tumors per animal, and the number of tumors per tumor-bearing animal induced by MAM were severalfold higher. This would suggest the presence of a gene(s) repressing metabolism of DMH to MAM. Moreover, differences in response to the carcinogens were observed between the sexes. In contrast to males, females treated with both DMH and MAM had significantly greater numbers of tumors per animal, tumors per tumor-bearing mice, and a greater proliferative response with extension of S-phase cells to the upper third and luminal surface of crypts. Among males, those with the XAKR/YSWR heritage appeared more resistant than XSWR/YAKR males, particularly in their response to MAM. A twofold difference in the number of foci of dysplasia per mouse, tumors per animal, and the number of tumors per tumor-bearing animals was seen. Analyses of the response to DMH and MAM by F1 reciprocal hybrids of the AKR and SWR strains have shown a complex inheritance pattern governing susceptibility to DMH. Resistance to the carcinogen is provided by at least two specific repressor genes, one governing metabolism of carcinogen from DMH to MAM, and the other controlled by gender. Genetic factors contributed by the AKR female appear to convey additional resistance to male progeny, suggesting more than one gender-related gene.

[The production of epidermal growth factor and polypeptides similar to it in the intestinal mucosa of rats in 1,2-dimethylhydrazine-induced carcinogenesis and compensatory regeneration]. / Produktsiia épidermal'nogo faktora rosta i podobnykh emu polipeptidov slizistoi obolochke kishechnika krys pri indutsirovannom 1,2-dimetilgidrazinom kantserogeneze i kompensatornoi regeneratsii.

The production of EGF and EGF-like polypeptides in the normal intestinal mucosa and during 1,2-dimethylhydrazine (DMH)-induced carcinogenesis and postresection regeneration was studied in albino rats using chromatographic separation of acid-ethanol extracts. Fractions after gel filtration on Biogel P-60 with subsequent reverse-phase high performance liquid chromatography in acetonitrile gradient were tested in radioreceptor assay for competition with EGF. It has been established that intestinal tumours induced by DMH and regenerating intestinal mucosa have amplified production of EGF--alpha-TGF and related proteins of high molecular weight (approx. 30; 45-55; 120 kD) with the EGF-competitive activity. It is supposed that the high molecular weight EGF-competitive material represents nonprocessed forms of EGF and/or alpha-TGF which are accumulated in rapidly proliferating low-differentiated cells due to the intensified expression of their genes.

1,2-Dimethylhydrazine-induced carcinogenesis influenced by different colonic anastomoses in rats.

Dispensing subcutaneously 1,2-dimethylhydrazine, intestinal carcinogenesis was investigated in male Fisher rats with different surgical colonic anastomoses, producing blind gut loops of isoperistaltic high (fecal stasis) and anisoperistaltic low fecal contact. One hundred and eight rats, except 1 rat of the control group, developed colonic neoplasms. In contrast to the control group, mainly in anastomotic areas and in isoperistaltic blind gut loops with intensive fecal contact huge adenocarcinomas of exophytic growth appeared with a mean tumor diameter (MTD) of 1.9 +/- 0.7 to 2.2 +/- 0.8 cm. Even in the control group, where rats only underwent laparotomy, we observed small polypoid adenocarcinomas mainly located in the distal colon (MTD: 0.7 +/- 0.3 cm). Anastomotic areas and isoperistaltic blind loops with intensive fecal contact proved to be regions with a higher risk for carcinoma formation.

Influence of dietary cis and trans fats on DMH-induced colon tumors, steroid excretion, and eicosanoid production in rats prone to colon cancer.

The effect of geometrical isomerism of dietary fats on colon tumorigenesis was studied in male and female rats of a strain prone to colon cancer (Wistar-Furth-Osaka). The rats were fed purified diets containing either partially hydrogenated corn oil (trans fat) or high-oleic safflower (cis fat) at the 5% level for one week and received a single oral dose of 1,2-dimethylhydrazine. The difference in the fatty acid composition of dietary fats was confined solely to the geometry of octadecenoate. An appropriate level of linoleic acid (2% of total energy) was supplied. After about 60 weeks, neither fat-type nor sex-dependent differences in the incidence of colon and small intestinal tumors was observed. The fecal excretion of neutral but not acidic steroids was higher in male rats fed the trans fat than in those fed the cis fat, but the composition remained almost unchanged. Aortic production of prostacyclin and the plasma concentration of thromboxane B2 were not influenced by dietary fats, although these were significantly higher in females, irregardless of the fat source. Thus, trans fat behaved much like the cis fat in various parameters, except for steroid excretion.