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Effect of a single dose of dimethylnitrosamine on biosynthesis of nucleic acid and protein in rat liver and kidney.

ABSTRACT: 1. Administration of a single dose of dimethylnitrosamine to rats temporarily fed on a protein-deficient diet causes a high incidence of kidney tumours. The effect of such a dose of dimethylnitrosamine (40mg/kg body wt.) on metabolism of nucleic acids and protein in rat liver and kidneys was examined during the week immediately after administration. 2. Incorporation of [(14)C]leucine and [(14)C]orotate into hepatic macromolecules was inhibited within 5h of injection of dimethylnitrosamine, and did not recover for at least 5 days. Interpretation of these results is complicated by the concomitant extensive hepatic necrosis. 3. Renal RNA synthesis was assayed by incorporation of [(14)C]orotate in vivo and measurement of DNA-dependent RNA polymerase activity in vitro. Both systems indicate biphasic inhibition; minimal activity was recorded 9h and 3 days after treatment. Changes in incorporation of [(14)C]leucine into renal protein were similar but less marked. 4. Sucrose-density-gradient analysis of renal cytoplasmic RNA indicated increased synthesis of rRNA 24h after injection of the nitrosamine. The rate of loss of radioactivity from kidney ribosomes pre-labelled with [(14)C]orotate was not modified by dimethylnitrosamine. 5. Dimethylnitrosamine increased incorporation of [(3)H]-thymidine into renal DNA. The three distinct periods of stimulated synthesis observed are discussed, with particular reference to recently published morphological studies of the sequential development of kidney tumours induced by dimethylnitrosamine in protein-depleted rats.

SUBMITTER: Stewart BW

PROVIDER: S-EPMC1178255 | biostudies-other | 1971 Dec

REPOSITORIES: biostudies-other

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Project description:Dimethylnitrosamine is metabolized to form an alkylating intermediate, which may have significance for its carcinogenic action. However, certain other compounds that are known to be highly mutagenic, including nitrous acid and hydroxylamine, might also be formed. Owing to the general reactivity of these compounds, it would be difficult to detect their formation in the intact animal. Instead, the nucleic acids of carcinogen-treated animals were examined for products of reaction with nitrous acid and hydroxylamine, i.e. for deamination of adenine and guanine, and formation of N(6)-hydroxycytosine, respectively. A double-labelling technique was used to detect very small amounts of the abnormal bases. The purine moieties of DNA in adult rat liver were labelled either with (14)C or with (3)H, by treating the neonatal animals with [(14)C]formate or with [(3)H]formate, and then allowing a period for normal growth. During this time, in liver, the labels were largely lost by metabolic turnover from cell components other than DNA. The pyrimidine moieties in DNA were labelled by treating the neonatal animals with [(14)C]orotate. The purine constituents of RNA of adult rat liver were labelled by repeated administration of [(14)C]- or [(3)H]-formate to the adult rats. The [(14)C]nucleic acid-labelled rat could then be treated with the carcinogen, and the [(3)H]nucleic acid-labelled animal could be used as a control. By this means the experimental and control tissues could be homogenized together in a single preparation, and the nucleic acids from the two tissues could be isolated, hydrolysed and analysed in a single sample. It was therefore possible to have an internal control for artifacts due to changes taking place in the nucleic acid bases during the experimental procedures. With this technique, the formation in vivo of 7-methylguanine in rat-liver DNA and RNA after administration of dimethylnitrosamine was confirmed, and no evidence was found for the formation of xanthine, hypoxanthine, N(6)-hydroxycytosine, or any other abnormal base.

Project description:1. The amounts of 7-methylguanine and O(6)-methylguanine present in the DNA of liver and kidney of rats 4h and 24h after administration of low doses of dimethylnitrosamine were measured. 2. O(6)-Methylguanine was rapidly removed from liver DNA so that less than 15% of the expected amount (on the basis of 7-methylguanine found) was present within 4h after doses of 0.25mg/kg body wt. or less. Within 24h of administration of dimethylnitrosamine at doses of 1mg/kg or below, more than 85% of the expected amount of O(6)-methylguanine was removed. Removal was most efficient (defined in terms of the percentage of the O(6)-methylguanine formed that was subsequently lost within 24h) after doses of 0.25-0.5mg/kg body wt. At doses greater or less than this the removal was less efficient, even though the absolute amount of O(6)-methylguanine lost during 24h increased with the dose of dimethylnitrosamine over the entire range of doses from 0.001 to 20mg/kg body wt. 3. Alkylation of kidney DNA after intraperitoneal injections of 1-50mug of dimethylnitrosamine/kg body wt. occurred at about one-tenth the extent of alkylation of liver DNA. Removal of O(6)-methylguanine from the DNA also took place in the kidney, but was slower than in the liver. 4. After oral administration of these doses of dimethylnitrosamine, the alkylation of kidney DNA was much less than after intraperitoneal administration and represented only 1-2% of that found in the liver. 5. Alkylation of liver and kidney DNA was readily detectable when measured 24h after the final injection in rats that received daily injections of 1mug of [(3)H]dimethylnitrosamine/kg for 2 or 3 weeks. After 3 weeks, O(6)-methylguanine contents in the liver DNA were about 1% of the 7-methylguanine contents. The amount of 7-methylguanine in the liver DNA was 10 times that in the kidney DNA, but liver O(6)-methylguanine contents were only twice those in the kidney. 6. Extracts able to catalyse the removal of O(6)-methylguanine from alkylated DNA in vitro were isolated from liver and kidney. These extracts did not lead to the loss of 7-methylguanine from DNA. 7. The possible relevance of the formation and removal of O(6)-methylguanine in DNA to the risk of tumour induction by exposure to low concentrations of dimethylnitrosamine is discussed.

Project description:BACKGROUND:Glyphosate-based herbicides (GBH) are the major pesticides used worldwide. Converging evidence suggests that GBH, such as Roundup, pose a particular health risk to liver and kidneys although low environmentally relevant doses have not been examined. To address this issue, a 2-year study in rats administering 0.1 ppb Roundup (50 ng/L glyphosate equivalent) via drinking water (giving a daily intake of 4 ng/kg bw/day of glyphosate) was conducted. A marked increased incidence of anatomorphological and blood/urine biochemical changes was indicative of liver and kidney structure and functional pathology. In order to confirm these findings we have conducted a transcriptome microarray analysis of the liver and kidneys from these same animals. RESULTS:The expression of 4224 and 4447 transcript clusters (a group of probes corresponding to a known or putative gene) were found to be altered respectively in liver and kidney (p < 0.01, q < 0.08). Changes in gene expression varied from -3.5 to 3.7 fold in liver and from -4.3 to 5.3 in kidneys. Among the 1319 transcript clusters whose expression was altered in both tissues, ontological enrichment in 3 functional categories among 868 genes were found. First, genes involved in mRNA splicing and small nucleolar RNA were mostly upregulated, suggesting disruption of normal spliceosome activity. Electron microscopic analysis of hepatocytes confirmed nucleolar structural disruption. Second, genes controlling chromatin structure (especially histone-lysine N-methyltransferases) were mostly upregulated. Third, genes related to respiratory chain complex I and the tricarboxylic acid cycle were mostly downregulated. Pathway analysis suggests a modulation of the mTOR and phosphatidylinositol signalling pathways. Gene disturbances associated with the chronic administration of ultra-low dose Roundup reflect a liver and kidney lipotoxic condition and increased cellular growth that may be linked with regeneration in response to toxic effects causing damage to tissues. Observed alterations in gene expression were consistent with fibrosis, necrosis, phospholipidosis, mitochondrial membrane dysfunction and ischemia, which correlate with and thus confirm observations of pathology made at an anatomical, histological and biochemical level. CONCLUSION:Our results suggest that chronic exposure to a GBH in an established laboratory animal toxicity model system at an ultra-low, environmental dose can result in liver and kidney damage with potential significant health implications for animal and human populations.

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Effect of a single dose of dimethylnitrosamine on biosynthesis of nucleic acid and protein in rat liver and kidney.

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