Effect of protein diet on age-dependent methylation of liver nuclear components of mice administered N-nitrosodimethylamine.

Protein diets deficient or enriched in DL-methionine were studied for the effect of methylation of nuclear components in subadult and adult outbred albino male mice given N-nitrosodimethylamine. After the mice were fed diets for 6 days, they were given ip injections of [14C]N-nitrosodimethylamine (5 mg/kg body wt) and killed 45 minutes later. Liver nuclei were isolated; incorporation of 14C into nuclear proteins and methylation of DNA and RNA were determined. In the subadult and adult animals the nuclear protein content was diminished by shortage of a single essential amino acid. Incorporation of 14C into the total protein was reduced. The reduction amounted to 55% in the subadult and 23% in the adult animals. Neither an age-dependent nor a diet-dependent change in the specific methylation of DNA and RNA was observed. Analysis of methylation of DNA and RNA was observed. Analysis of methylated purine bases showed an age-related rise in O6-methylguanine in the adult as compared with the subadult mice.

Novel Chinese hamster ultraviolet-sensitive mutants for excision repair form complementation groups 9 and 10.

In this paper we demonstrate that the mutants CHO7PV and CHO4PV isolated by us from the CHO-K1 prol- cell line represent two new complementation groups of UV-sensitive excision repair-defective rodent mutants. We have classified the mutant CHO7PV as representative of Group 9 and CHO4PV as representative of Group 10. Cellular and biochemical characterization of these mutants indicates that they are moderately sensitive to a broad spectrum of mutagens (UV and mono- and bifunctional alkylating agents), partially unable to perform UV-induced DNA repair synthesis, and partially defective in the incision step of the DNA excision repair pathway and in the removal of the two main lesions caused by UV [cyclobutane pyrimidine dimers and (6-4) photo-products]. In terms of UV survival and incision, CHO4PV is apparently more defective than CHO7PV (40% and 50% of wild-type survival, respectively, and 55% and 75% of wild-type incision), whereas when repair DNA synthesis and lesion removal are compared, CHO7PV seems to be more severely affected (30% of wild-type unscheduled DNA synthesis in CHO7PV and 60% in CHO4PV). This suggests a subtlety in the relation between removal of these specific lesions and overall repair capacity and survival.

Protein synthesis activity in vitro in mouse tissues in relation to methionine-cysteine deficiency and age.

The objective of this study was to determine whether ageing in the mouse is associated with changes in protein synthesis activity and response to dietary methionine-cysteine deficiency. Growing subadult mice (30-day-old) and adult mice (70-day-old) were compared. The amino acid restricted diet contained methionine and cysteine at a level of 10% that of the control diet. After a feeding period of 6 days protein synthesis activity was measured in liver, kidney and lung. In control liver, the level of protein synthesis activity was the same for the two age groups; in kidney and lung, activity was diminished in the adult group. After methionine-cysteine deficiency protein synthesis activity was unchanged in the subadult and diminished in the adult liver. In kidney, both age groups responded with an increase in activity. In lung, the subadult group remained unchanged and activity in the adult group was elevated. The amount of DNA/g wet weight of tissue was higher in the subadult than the adult group throughout. Methionine-cysteine deficiency resulted in an increase in DNA/g tissue wet weight, while the total amount of DNA in liver decreased, but was unchanged in kidney and lung. Total liver activity was decreased less in subadult than adult mice. Total kidney and lung activities were diminished in subadult but not adult mice. The results suggest that the specific protein synthesis activity of the ribosomes in the kidney and lung tends to decrease with age more readily than in liver, and that subadult liver displays less sensitivity towards methionine-cysteine deficiency than does adult liver.

Methionine-cysteine deficiency and alkylation of DNA in liver, kidney and lung of mice administered dimethylnitrosamine.

The effect of methionine-cysteine deficiency on the methylation of DNA purines by dimethylnitrosamine metabolites was studied in subadult and adult mice. In liver, no dietary effect on the specific methylation of 7-methylguanine was observed, while that of 3-methyladenine decreased in the adult animals. The specific methylation of guanine in the 0(6)-position and the ratio of 0(6)-methylguanine to 7-methylguanine increased significantly after methionine-cysteine deficiency. Methylation in kidney decreased in subadult but increased in adult mice. In lung, the amount of 7-methylguanine was significantly elevated after methionine-cysteine deficiency in both the subadult and adult mice. The results demonstrate an increase in the specific methylation in liver of guanine in the 0(6)-position by the methionine-cysteine deficient diet, together with differences in the methylation pattern between organs of the two age groups.

Contractile protein breakdown in human leg skeletal muscle as estimated by [2H3]-3-methylhistidine: a new method.

3-Methylhistidine urinary excretion and net balances across the leg or forearm have been used as markers of contractile protein breakdown in muscle tissue. Here we investigate whether infusion of labeled 3-methylhistidine and the measurement of the arteriovenous dilution of the tracer with unlabeled 3-methylhistidine will result in more consistent and precise measurements of 3-methylhistidine rates of appearance and consequently muscle contractile protein breakdown rates in comparison with conventional arteriovenous concentration difference measurements. Six healthy volunteers were studied in the postabsorptive state and received a primed continuous infusion of 3-[2H3-methyl]- methylhistidine and L-[ring-2H5]-phenylalanine for 4 hours. 2H3-3-methylhistidine reached an isotopic steady state after 210 minutes in all subjects. Arteriovenous differences of 3-methylhistidine, measured by high-performance liquid chromatography (HPLC), showed both uptake and release from skeletal muscle, which is theoretically not likely to occur. The enrichment of 2H3-3-methylhistidine was consistently lower in the femoral vein than in the artery, and therefore a constant net release of 3-methylhistidine from the leg was observed. The mean rates of appearance for 3-methylhistidine and phenylalanine were 0.44 +/- 0.30 nmol x min(-1) x 100 mL(-1) and 11.2 +/- 5.7 nmol x min(-1) x 100 mL(-1), respectively. In summary, arteriovenous difference measurement of 2H3-3-methylhistidine enrichment is more reliable than measurement of arteriovenous difference of unlabeled 3-methylhistidine. Consequently, measuring rates of appearance from leg muscle using labeled 3-methylhistidine resulted in more consistent and accurate values of contractile protein degradation rates in human skeletal muscle.

Longitudinal pattern of glutamine/glutamate balance across the leg in long-stay intensive care unit patients.

BACKGROUND & AIMS: Progressive muscle wasting is a characteristic feature of patients treated at the intensive care unit (ICU). As a consequence, endogenous glutamine production by skeletal muscle may be compromised. We investigated the time pattern of the glutamine and glutamate net balance across the leg in long-stay ICU patients. METHODS: Critically ill patients with multiple organ failure that were expected to stay in the ICU for more than 3 days were included in a longitudinal study. Possible changes in amino acid net balance over the leg muscle were investigated overtime. The patients (n=20) were studied descriptively every third or fourth day, on a total of 2-7 occasions. MAIN RESULTS: The glutamine net release from leg muscles did not change significantly during the initial 2 weeks of ICU stay and was not related to the plasma concentration of glutamine. The net uptake of glutamate across the leg muscles was unaltered during this time period, but it was found to correlate statistically with both the arterial glutamate concentration and the glutamine net release. A continuous net release of phenylalanine indicated a progressive net loss of muscle protein in these patients. CONCLUSION: The net release of glutamine from skeletal muscle does not decrease in stabilized critically ill patients with multiple organ failure over the initial 2 weeks of ICU stay, despite progressive muscle wasting.

DNA damage and repair after low doses of UV-C radiation; comparable rates of repair in rodent and human cells.

The UV endonuclease assay for cyclobutane pyrimidine dimers in the DNA of UV-C-irradiated mammalian cells has been modified by replacing alkaline sucrose sedimentation with alkaline unwinding and hydroxylapatite chromatography to determine the number of DNA breaks introduced by the endonuclease. Dimers induced by doses as low as 0.25 Jm-2 can be detected and the assay has been used to examine the capacity of human, hamster and mouse cells to remove damage inflicted by sublethal doses of UV-C. In addition, incision activity has been measured by incubating cells with DNA synthesis inhibitors after irradiation with UV-C. In rodent and human cells, given a dose of UV-C of 1 Jm-2 about half of the endonuclease-sensitive sites are lost in 5-6 h. The incision capacity of these cells corresponds well with the extent of removal of dimers. Thus, although rodent cells are normally considered to be relatively deficient in nucleotide excision repair, we find that rodent and human cells have comparable capacities to deal with low levels of UV-C-induced damage.

O6-methylguanine-DNA methyltransferase and alkylation of liver DNA in mice exposed to dimethylnitrosamine during dietary deficiency of essential amino acids.

Male NMRI mice were fed a diet containing a complete mixture of amino acids or a mixture deficient in methionine-cysteine or lysine (30% of the control level) for a period of 6 days. During the feeding period all mice received dimethylnitrosamine in the drinking water ad libitum. The exposure averaged 1 mg dimethylnitrosamine/kg body weight and day. The concentration of O6-methylguanine-DNA methyltransferase was measured in liver extracts. It decreased significantly in the methionine-cysteine deficient mice. When DNA from the liver was analyzed for alkylated purine bases the mice received a single dose of 14C-labeled dimethylnitrosamine (0.5 or 1 mg/kg body weight) at 120 min before sacrifice. The concentration of O6-methylguanine increased significantly over the control level upon feeding the deficient diets and was restored to the concentration of the controls by refeeding lysine for 2 days following 6 days of lysine deficiency. The increased ratio of O6-methylguanine to N-7-methylguanine indicated that methylation of guanine in the N-7 position was not subject to variation by the intake of dimethylnitrosamine during the dietary deficiencies. The results demonstrate the requirement for a balanced composition of amino acids in the diet to maintain a sufficient concentration of O6-methylguanine-DNA methyltransferase in the cells and thus to permit efficient removal of the methyl group from the O-6 position of guanine in DNA after exposure to dimethylnitrosamine.

The comet assay: mechanisms and technical considerations.

The comet assay is frequently used to measure DNA damage in individual cells. In order to better understand the mechanisms behind the technique, we have studied the behaviour of DNA under different electrophoresis conditions in mammalian cells exposed to gamma radiation. The comet tails obtained after neutral electrophoresis seem to consist of DNA loops which are attached to structures in the nucleus, since the DNA cannot move in the second direction after two-dimensional electrophoresis. When the DNA is labelled by a short pulse, microautoradiography reveals that all label appear in the head of the comets when neutral electrophoresis is applied. After chase incubation, the label moves out into the tails. This gives further support to the view that the DNA loops are fixed to some structure in the nucleus where also the DNA synthesis takes place. Under alkaline electrophoresis conditions, however, the entire comet tails move in the new electrophoresis direction. Thus, it appears that the alkaline comet tails consist of free DNA fragments. Further, the effects of alkaline concentration and sodium chloride during unwinding and electrophoresis are discussed. Throughout the study, a protocol for drying and fixation of the comets has been used.

Combined effect of dimethylnitrosamine and a lysine-restricted diet on O6-methylguanine-DNA methyltransferase levels in mouse tissues.

O6-Methylguanine is a lesion produced in DNA after exposure of animals to the procarcinogen dimethylnitrosamine. The lesion may lead to mutagenesis or carcinogenesis if not repaired. Repair is accomplished by the protein O6-methylguanine-DNA methyltransferase (MT). The methyl group is transferred to a cysteine residue of the protein, which is not regenerated. In mice, after exposure to alkylating agents, the synthesis of the protein is non-inducible. The inactivation of MT as a result of the transmethylation makes new synthesis of the protein molecules necessary for further dealkylation reactions. Protein synthesis activity correlates well with dietary protein quality. Nutritional conditions of amino acid restriction will limit the number of MT molecules synthesized. Continuous exposure of mice to dimethylnitrosamine will further diminish the pool of the protein. In this study, mice were fed a diet low in lysine and simultaneously given dimethylnitrosamine in the drinking water at concentrations resulting in dosages of zero, 0.4 mg or 1.2 mg/kg body weight/day. After 6 days MT was assayed in liver, kidney and lung. In liver and kidney, lysine restriction provoked a decrease in MT levels per mg of tissue DNA which was intensified by the presence of dimethylnitrosamine in the drinking water. Recovery from lysine restriction with respect to MT levels was achieved within 2 days. In lung, moderate effects on MT levels were observed when dietary lysine restriction was combined with the highest dosage of dimethylnitrosamine used (1.2 mg/kg body weight/day). The data strongly emphasize the importance of an adequate amino acid mixture in the diet, to support protein synthesis and to allow for high MT levels and repair of DNA lesions at the O-6 position of guanine during the exposure of the animals to alkylating agents.

Diet- and age-dependent modulation of O6-methylguanine-DNA methyltransferase levels in mouse tissues.

Methylation of guanine in the O6 position by N-nitrosodimethylamine is higher in liver DNA of mice fed a diet restricted in essential amino acids than in controls. A diminished content of the repair enzyme O6-methylguanine-DNA methyltransferase (AAT) may be responsible for the elevated level of O6-methylguanine observed after amino acid deficiency. A delayed repair of O6-methylguanine can be ascribed to diminished AAT activity in amino-acid-restricted mice. In adults, but not in subadults, the diminished AAT activity in liver correlated well with a 40% decrease in overall protein synthesis.

O6-methylguanine-DNA methyltransferase levels in tissues of methionine-cysteine deficient subadult and adult mice.

1. Subadult and adult mice were fed during 6 days a diet containing a complete mixture of amino acids or a mixture low in methionine-cysteine. 2. O6-methylguanine-DNA methyltransferase (MT) is the acceptor protein for methyl groups present in DNA at the O-6 position of a guanine that has been methylated by alkylating carcinogenic agents. 3. Upon methionine-cysteine deficiency O6-methylguanine-DNA methyltransferase levels decreased significantly in liver, but seemed unaffected in kidney, lung, testis and brain. 4. Age associated differences were found in liver, with lower values in the subadult than the adult mice leaving the young animals more vulnerable to exposure to alkylating agents. 5. To ensure an efficient repair of DNA lesions both age groups depended on a complete amino acid mixture in the diet.

Non-random effect on RNA synthesis in liver chromatin by administration of dimethylnitrosamine to mice.

The effect of dimethylnitrosamine on functional activities of liver chromatin was studied in mice. After a single dose of dimethylnitrosamine injected i.v. (25 mg/kg body wt, 45 min before sacrifice) liver nuclei were isolated and incubated with micrococcal nuclease (EC 3.1.4.7) to an acid-solubility of 2.5% of total DNA. Chromatin was fractionated into a 1,200 g pellet P1, 102,000 g pellet P2 and supernatant fraction S2. Chromatin-bound RNA polymerase I plus III activity decreased 15% in the P1 and 25% in the P2 fraction. No changes in activity were observed in the S2 fraction. Chromatin-bound RNA polymerase II activity decreased 19% in the P1, 49% in the P2 and 32% in the S2 fraction. Heparin stimulated RNA polymerase II activity decreased 10% in the P1 and 44% in the P2 fraction. Formation of initiation in nuclear lysates with RNA polymerase from Escherichia coli increased after administration of dimethylnitrosamine suggesting an increase in the number of sites available for the start of new RNA chains. The results show that limited digestion of nuclei with endonuclease cleaves chromatin regions which are more affected by dimethylnitrosamine than the total chromatin suggesting a non-random effect of the hepatotoxin on chromatin. Modifications of the DNA template by dimethylnitrosamine is indicated by the change in number of initiation complexes.

In vivo distribution of dimethylnitrosamine metabolites in mouse liver and their binding to micrococcal nuclease sensitive and resistant chromatin.

Mice were injected i.p. with a single dose (5 mg/kg body wt) of [14C]dimethylnitrosamine and killed at time intervals between 15 and 120 min. Isolated nuclei were incubated with micrococcal nuclease and the chromatin separated into a 1100 g pellet P1 and supernatant fraction S1. The incorporation of 14C from [14C]dimethylnitrosamine into chromatin was significantly higher in the P1 than the S1 fraction. The purine bases of the P1 DNA showed a lower methylation than those of the S1 DNA. In contrast, radioactivity of the proteins was higher in the P1 than the S1 fraction. It is concluded that the open structures of the S1 chromatin were preferentially attacked by the hepatotoxin leading to a high 14C-labelling of the DNA relative to that of the proteins.

O6-methylguanine-DNA methyltransferase content in human lymphocytes following surgical trauma.

O6-methylguanine-DNA methyltransferase removes methyl groups from the O-6 position of guanine in DNA previously alkylated by alkylating carcinogens. Thus, the protein facilitates restoration of the impaired DNA. The content of O6-methylguanine-DNA methyltransferase was assayed in circulating lymphocytes and the impact of surgical trauma investigated. Patients (n = 13) without metabolic diseases admitted for elective orthopedic surgery were used. The patients were allowed water and food postoperatively. Blood was taken before and 3 days following surgery and the circulating lymphocytes were isolated. Before surgery, the O6-methylguanine-DNA methyltransferase content determined in the cell extracts showed patient-specific variations. Following surgery, a significant decrease of the protein by 60% (from 609 to 243 fmole/mg of DNA) was observed. The intensity of surgical trauma was confirmed by the decrease in plasma albumin concentration and the increase in white blood cell counts. The surgical trauma might elicit its effect as either a change in turnover of O6-methylguanine-DNA methyltransferase or a release from the thymus of lymphocytes low in enzyme levels. In summary, the surgical trauma per se was the cause of the pronounced decrease in the O6-methylguanine-DNA methyltransferase seen here. Investigations on O6-methylguanine-DNA methyltransferase levels have an important relevance in studies on tumor-promoting agents inhaled and then taken up by the T lymphocytes of prospective proliferating capacity.