The stability of rat liver ribonucleic acid in vivo after methylation with methyl methanesulphonate or dimethylnitrosamine

1. RNA was isolated from rat liver at selected times after the intraperitoneal injection of either [¹⁴C]methyl methanesulphonate (50mg/kg) or [¹⁴C]dimethylnitrosamine (2mg/kg). These doses were chosen to minimize effects due to toxicity. 2. Two methods of extraction and purification of RNA were used and an analysis of the radioactivity present was made by column chromatography of acid hydrolysates of the purified RNA. 3. The extent of methylation of guanine, the principal site of alkylation in rat liver RNA, was determined at times up to 14 days after injection. Although dimethylnitrosamine is a potent liver carcinogen and methyl methanesulphonate is not carcinogenic to rat liver, the rate of disappearance of 7-methylguanine from RNA was similar for both compounds, with a half-life of about 3.5 days. 4. An estimate of the biological half-life of rRNA was made by using [³H]orotic acid. A half-life of 5 days was obtained and this was not affected by injecting animals with unlabelled methyl methanesulphonate at the same dosage of 50mg/kg used in the studies of RNA methylation. 5. After administration of labelled orotic acid, reutilization of labelled RNA degradation products probably results in an overestimation of the biological half-life for rRNA. It is suggested that non-toxic doses of methylating agents such as methyl methanesulphonate and dimethylnitrosamine may prove to be a more effective way of accurately estimating the biological turnover of RNA species.     

Formation and subsequent removal of O6-methylguanine from deoxyribonucleic acid in rat liver and kidney after small doses of dimethylnitrosamine

1. The amounts of 7-methylguanine and O⁶-methylguanine present in the DNA of liver and kidney of rats 4h and 24h after administration of low doses of dimethylnitrosamine were measured. 2. O⁶-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 O⁶-methylguanine was removed. Removal was most efficient (defined in terms of the percentage of the O⁶-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 O⁶-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 1–50μg of dimethylnitrosamine/kg body wt. occurred at about one-tenth the extent of alkylation of liver DNA. Removal of O⁶-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 1μg of [³H]dimethylnitrosamine/kg for 2 or 3 weeks. After 3 weeks, O⁶-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 O⁶-methylguanine contents were only twice those in the kidney. 6. Extracts able to catalyse the removal of O⁶-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 O⁶-methylguanine in DNA to the risk of tumour induction by exposure to low concentrations of dimethylnitrosamine is discussed.     

Methylated purines in the deoxyribonucleic acid of various Syrian-golden-hamster tissues after administration of a hepatocarcinogenic dose of dimethylnitrosamine.

1. DNA was extracted from livers, kidneys and lungs of Syrian golden hamsters at various times (up to 96h) after injection of a hepatocarcinogenic dose of [14C]dimethylnitrosamine. Purine bases were released from the DNA by mild acid hydrolysis and separated by Sephadex G-10 chromatography. 2. At 7h after dimethylnitrosamine administration liver DNA was alkylated to the greatest extent, followed by that of lung and kidney, the values for which were 8 and 3% respectively of those for liver. 3. The O6-methylguanine/7-methylguanine ratios were initially the same in all three organs and in the liver DNA of rats under similar conditions of dose. 4. O6-Methylguanine was the most persistent alkylated purine in all three hamster tissues. There was evidence for excision of 7-methyl-guanine, the highest activity for this being present in the liver. 5. Detectable amounts of the minor products 3-methyladenine, 1-methyladenine, 3-methylguanine and 7-methyladenine were present in most hamster tissues, and their individual rates of loss from liver DNA were determined. 6. Ring-labelling of the normal purines in DNA was highest in the liver, followed closely by the lung (80% of that in liver) whereas the kidney had very low incorporation (3% of that in liver). 7. The results are discussed with respect to the hepatotoxicity of dimethylnitrosamine, the miscoding potential of the various alkylation products and the induction of liver tumours in hamsters.     

Alkylation of deoxyribonucleic acid in vivo in various organs of C57BL mice by the carcinogens N-methyl-N-nitrosourea, N-ethyl-N-nitrosourea and ethyl methanesulphonate in relation to induction of thymic lymphoma. Some applications of high-pressure liquid chromatography.

1. Methods were developed for analysis of alkylpurines, O2-alkylcytosines, and representative phosphotriesters [alkyl derivatives of thymidylyl(3'-5')thymidine], in DNA alkylated in vivo, using high-pressure liquid chromatography. 2. The patterns of alkylation products in DNA in vivo at short times were closely similar to those found for reactions in vitro. Alkylation by the nitrosoureas was complete in vivo within 1 h, but with ethyl methanesulphonate was maximal at 2--4h. 3. The time course of persistence of alkylation products in vivo was determined for several tissues. In addition to the rapid loss of 3- and 7-alkyladenines reported previously for all tissues, a relatively rapid loss of O6-alkylguanines from DNA of liver was found which was more rapid at lower doses. In brain, lung and kidney, excision of O6-alkylguanine was much less marked, but was not entirely excluded by the data. In thymus, bone marrow and small bowel, all alkylated bases were lost with half-lives of 12--24h, at non-cytotoxic doses of alkylation. 4. No evidence for any marked excision of other minor products from alkylated DNA in vivo was found; thus 1-methyladenine, O2-ethylcytosine (found in appreciable amount only with N-ethyl-N-nitrosourea), 3-methylguanine, and dTp(Alk)dT persisted in alkylated DNA, including DNA of liver. 5. The induction of thymic lymphoma was determined over the range of single doses by intraperitoneal injection up to about 60% of the LD50 values, and related to the extent of alkylation of target tissues thymus and bone marrow. With N-methyl-N-nitrosourea over 90% tumour yield was attained at 60 mg/kg, and with N-ethyl-N-nitrosourea up to 52% at 240 mg/kg, but with ethyl methanesulphonate at up to 400 mg/kg only a few per cent of tumours were obtained. 6. The carcinogenic effectiveness of the agents was positively correlated with the extents of alkylation of guanine in DNA of target tissues at the O-6 atom. On the basis that at doses giving equal carcinogenic response these extents of alkylation would be equal, the chemical analyses showed that the ratio of equipotent doses to that for N-methyl-N-nitrosourea would be, for N-ethyl-N-nitrosourea, 5.3 for ethyl methanesulphonate about 21, and for methyl methanesulphonate [Frei & Lawley (1976) Chem.-Biol. Interact. 13, 215--222] about 144. These predictions were in reasonably good agreement with the observed dose-response data for these agents.     

Synthesis and pregnancy-inhibiting activity of 7-substituted androst-5-ene derivatives.

Synthesis of 7-aryl/allyl-substituted androstene derivatives 3a through 3g has been carried out by Grignard reaction on 3 beta,17 beta-diacetoxyandrost-5-en-7-one (2) with aryl/allyl magnesium bromide. Isomeric mixture of products 3b and 3c/3e and 3f/3h was separated by column chromatography. Stereochemical assignment at C-7 has been made on the basis of 13C nuclear magnetic resonance studies and chemical considerations. Compounds 6a and 6b were synthesized by alkylation of compound 5 with beta-(N,N-diethylamino)ethyl chloride hydrochloride and 1-(2-chloroethyl)pyrrolidine hydrochloride, respectively. Compound 3g (isomeric mixture) prevented pregnancy in 60% of rats at 10 mg/kg daily dose administered orally on days 1 to 7 of pregnancy; however, its only isolable 7 beta-hydroxy isomer, 3h, was inactive at this dose.     

Effect of cycloheximide on dimethylnitrosamine-induced polyribosome disaggregation in rat liver

The simultaneous administration of a dose of 1.5 mg/kg body wt. cycloheximide with 20 mg/kg body wt. dimethylnitrosamine to rats did not affect the metabolism of the nitrosamine as deduced by following its concentration in the blood nor affect the level of alkylation by the nitrosamine of cytoplasmic RNA in the liver. Incorporation of [¹⁴C]leucine into hepatic protein, which was maximally inhibited 60% 3 h after administration of the same dose of dimethylnitrosamine alone, was reduced by 94% within 1 h in rats treated with dimethylnitrosamine and cycloheximide.Polyribosome structure was determined by sucrose gradient centrifugation. Disaggregation of hepatic polyribosomes as a result of administration of the nitrosamine alone was most marked at 4 h, but by 8 h there was a recovery of polyribosome structure and a relative decrease in the number of monomeric ribosomes. Administration of cycloheximide alone did not affect the structure of hepatic polyribosomes. When dimethylnitrosamine and cycloheximide were given simultaneously the immediate breakdown of polyribosomes that normally followed administration of dimethylnitrosamine was prevented for at least 4 h; however after 8 h there was considerable disaggregation of the polyribosomes in the liver. The implications of these observations for the mechanism of inhibition of protein synthesis by dimethylnitrosamine are discussed.     

Adenine N3 is a main alkylation site of styrene oxide in double-stranded DNA

Styrene 7,8-oxide (SO), a major metabolite of styrene, is classified as a probable human carcinogen. In the present work, salmon testis DNA was reacted with SO and the alkylation products were analysed after sequential depurination in neutral or acidic conditions followed by HPLC separation and UV-detection. A novel finding was that the N-3 position of adenine was the next most reactive alkylation site in double-stranded DNA, comprising 4% of the total alkylation, as compared to alkylation at the N-7 position of guanine, 93% of the total alkylation. Both alpha- and beta-products of SO were formed at these two sites. Other modified sites were N2-guanine (1.5%, alpha-isomer), 1-adenine (0.4%, both isomers) and N6-adenine (0.7%, both isomers) as well as 1-hypoxanthine (0.1%, alpha-isomer), formed by deamination of the corresponding 1-adenine adduct. The results indicated that in double-stranded DNA N-7 of guanine and N-3 of adenine account for 97% of alkylation by SO. However, these abundant adducts are not stable, the half-life of depurination in DNA for 3-substituted adenines being approximately 10 and approximately 20 h, for alpha- and beta-isomers, respectively, and 51 h for both isomers of 7-substituted guanines.     

Efficacy and safety of artemether in the treatment of chronic fascioliasis in Egypt: exploratory phase-2 trials

Background

Fascioliasis is an emerging zoonotic disease of considerable veterinary and public health importance. Triclabendazole is the only available drug for treatment. Laboratory studies have documented promising fasciocidal properties of the artemisinins (e.g., artemether).

Methodology

We carried out two exploratory phase-2 trials to assess the efficacy and safety of oral artemether administered at (i) 6×80 mg over 3 consecutive days, and (ii) 3×200 mg within 24 h in 36 Fasciola-infected individuals in Egypt. Efficacy was determined by cure rate (CR) and egg reduction rate (ERR) based on multiple Kato-Katz thick smears before and after drug administration. Patients who remained Fasciola-positive following artemether dosing were treated with single 10 mg/kg oral triclabendazole. In case of treatment failure, triclabendazole was re-administered at 20 mg/kg in two divided doses.

Principal Findings

CRs achieved with 6×80 mg and 3×200 mg artemether were 35% and 6%, respectively. The corresponding ERRs were 63% and nil, respectively. Artemether was well tolerated. A high efficacy was observed with triclabendazole administered at 10 mg/kg (16 patients; CR: 67%, ERR: 94%) and 20 mg/kg (4 patients; CR: 75%, ERR: 96%).

Conclusions/Significance

Artemether, administered at malaria treatment regimens, shows no or only little effect against fascioliasis, and hence does not represent an alternative to triclabendazole. The role of artemether and other artemisinin derivatives as partner drug in combination chemotherapy remains to be elucidated.     

致癌剂DEN、促癌剂巴豆油对大鼠肝α_1抑制因子3基因表达的影响

本文对致癌剂二乙基亚硝胺(DEN)和促癌剂巴豆油对大鼠肝α_1-抑制因子3(α_1- I_s)基因表达的影响进行了观察。结果表明:在腹腔注射DEN(200mg/kg·b.w.)后2h,α_1- I_3 RNA水平即显著下降;4h左右有所回升,约为正常水平的四分之一;然后又下降至极低水平,并一直保持该水平至24h。同时,c-myc RNA在2h则被诱导,约为正常水平的2-3倍;之后,即在4至24h内虽有波动,但基本处于正常水平。在腹腔注射巴豆油(18mg/kg.b.w.)后1h,α_1-I_3RNA水平即下降,约为正常水平的1/8,此后有所回升,约为正常水平的1/3-1/4;在同样条件下,ODC和C-fos RNA能被巴豆油诱导而增加。我们以前的工作表明,在大多数(75％,12/16)由DEN所诱发的大鼠肝癌中,α_1- I_3RNA水平显著下降,且基因结构发生了变化。综合这些结果,说明α_1- I_3基因或与该基因表达有关的蛋白调控因子可能是致癌剂DEN或促癌剂TPA的作用靶分子。该基因表达异常的意义及机理正在深入研究中。     

ALKYLATION OF RAT BRAIN NUCLEIC ACIDS BY N-METHYL-N-NITROSOUREA AND METHYL METHANESULPHONATE

Abstract— Alkylation of rat brain nucleic acids in vivo was measured after a single intravenous injection (1 mmol/kg body wt.) of N -[¹⁴C]methyl- N -nitrosourea and [¹⁴C]methyl methanesulphonate. The main product with both compounds was 7-methylguanine, The extents of methylation on this position in DNA and RNA were similar with methylnitrosourea but methyl methanesulphonate produced twice as much 7-methylguanine in DNA as in cytoplasmic RNA. Brain DNA from rats treated with labelled methylnitrosourea contained radioactive O ⁶-methylguanine, accounting for about 12 per cent of the radioactivity present as 7-methylguanine and cytoplasmic RNA contained about half this amount of O ⁶-methylguanine. Neither DNA nor cytoplasmic RNA from methyl methanesulphonatetreated rats contained any detectable O ⁶-methylguanine. Treatment with both compounds resulted in varying small amounts of methylation of other nucleic acid bases including 1-methyladenine, 3-methyladenine and 3-methylcytosine. The possible relevance of alkylation of brain nucleic acids to the induction of brain tumours is discussed.     

Effects of haloperidol on rat behavior and density of dopaminergic D2-like receptors

The present work shows the effects of a typical neuroleptic drug (haloperidol, HAL) on rat behavior (catalepsy and locomotor activity) and dopaminergic D2-like receptor densities in the hippocampus and striatum. Male Wistar rats (2-3 months old) were treated daily for 30 days with HAL (0.2 or 1mg/kg, intraperitoneally (i.p.)). At the end of treatment and 1h or 1, 3, 7 and 15 days after drug withdrawal, animals were subjected to behavioral tests and sacrificed afterwards for binding assays. The results showed that behavioral effects with both doses were significant only 1h and 1 day after withdrawal, and similar to controls at the third day. An up-regulation of D2 receptors was observed in the striatum (28% increase) but not in the hippocampus after 24h HAL (1mg/kg) withdrawal. However, an up-regulation was seen in both areas (1mg/kg) 3 days after drug withdrawal (58 and 42% increases in the hippocampus and striatum, respectively), and continued after 7 days of withdrawal only in the striatum (43 and 49% for the doses of 0.2 and 1mg/kg, respectively), suggesting the influence of dose, age, and time of drug withdrawal on these parameters. The up-regulation disappeared after 15 days of haloperidol withdrawal. Increases (72 and 140%) in constant dissociation values (K(d)) values were also observed 7 days after withdrawal. Results show differences on a time-basis between behavioral alterations and dopaminergic D2 receptors up-regulation.     

In vivo repair of rat liver DNA damaged by dimethylnitrosamine or diethylnitrosamine.

Effects of hepatocarcinogens dimethylnitrosamine (DMN) and diethylnitrosamine (DEN) on the sedimentation pattern of rat liver DNA in alkaline sucrose gradients were studied with regard to time and dose dependency. Both DMN (10 mg/kg body weight) and den (13.4 or 134 mg/kg) induced appreciably decreased DNA sedimentation rates at 24 h after injection. DMN at 10 mg/kg was as effective in decreasing the DNA sedimentation rate at 24 h after injection as was the higher dose of DEN (134 mg/kg). Sedimentation patterns at 1, 6 and 14 days after injection indicated that damage induced by DEN (134 mg/kg) was repaired at a substantially lower rate than DMN (10 mg/kg) induced damage. When effects of equimolar doses of DMN (10 mg/kg) and DEN (13.4 mg/kg) were compared at 1, 6 and 14 days after injection, it was observed that the more pronounced damage of rat liver DNA induced by DMN was repaired at a faster rate than was the DEN-induced damage. At the molecular level this difference in repair between damage induced by the two nitrosamines is probably related to different DNA alkylation patterns. The relatively persistent nitrosamine-induced DNA lesions (observed especially after DEN administration) are thought to represent phosphotriesters which give rise to single strand DNA breaks at strongly alkaline conditions of lysis on top of the gradient. The results are discussed in relation to the possible significance of alkylation and repair of DNA in the formation of (pre)cancerous lesions in rat liver.     

In vivo interactions of acrylonitrile with macromolecules in rats

The irreversible binding of [2,3-14C]acrylonitrile (VCN) to proteins, RNA and DNA of various tissues of male Sprague-Dawley rats after a single oral dose of 46.5 mg/kg (0.5 LD50) has been studied. Proteins were isolated by chloroform-isoamyl alcohol-phenol extraction. RNA and DNA were separated by hydroxyapatite chromatography. Binding of VCN to proteins was extensive and was time dependent. Radioactivity in nucleic acids was registered in the liver and the target organs, stomach and brain. DNA alkylation, which increased by time, was significantly higher in the target organs, brain and stomach (119 and 81 pmol/mg, respectively, at 24 h) than that in the liver. The covalent binding indices for the liver, stomach and brain at 24 h after dosing were, 5.9, 51.9 and 65.3, respectively. These results suggest that VCN is able to act as a multipotent carcinogen by alkylation of DNA in the extrahepatic target tissues, stomach and brain.     

Optimum time for administration of indomethacin to inhibit ovulation in the rabbit

The antiinflammatory agent, indomethacin, inhibits ovulation in mammals by interfering with the synthesis of prostaglandins in preovulatory follicles. To determine the optimum time to administer this inhibitor, indomethacin was given at specific intervals from 10 h before, and up to 9 h after, the ovulatory process had been initiated by hCG (50 I.U./kg). The drug dosage ranged from 1.25 mg/kg to 40 mg/kg. The optimum time to give indomethacin was at 7–8 h after hCG (i.e., 2–3 h before expected rupture of the follicle) at which time the minimum effective dose was 2.5 mg/kg. Since a significant elevation in prostaglandin synthesis occurs as early as 3–5 h after hCG stimulation of rabbit follicles (1), these results reveal that nonsteroidal antiinflammatory agents can interrupt the ovulatory process even the follicle has begun producing substantial amounts of prostaglandins. The data suggest that prostaglandins need to be produced continuouly in the follicle up to the time of actual rupture, or else that indomethacin is interfering with some other aspect of the ovulatory process which transpires after the elevation of prostaglandins.     

Detection of micronuclei in peripheral blood of mitomycin C-treated mice using supravital staining with acridine orange.

The induction of micronuclei in peripheral blood from mitomycin C (MMC)-treated mice was examined using a supravital acridine orange staining method. Male ICR mice were intraperitoneally given MMC at a single dose of 0.25, 0.5, 1, or 2 mg/kg. Blood was sampled from the tail 24, 48, 72, and 96 h after treatment, and the frequency of micronucleated reticulocytes (MNRETs) was examined. The induction of MNRETs peaked at 48 h after treatment with MMC; there was a clear, dose-related increase in MNRETs. In a multiple-treatment study, mice were treated with 4 consecutive daily injections of MMC at a dose of 0.13, 0.25, 0.5, or 1 mg/kg. The frequency of MNRETs increased markedly 24 h after the second treatment as compared with the first treatment, and did not change significantly until 24 h after the fourth treatment. The frequency of MNRETs decreased to approximately control values 96 h after the last treatment. In addition, a slight but statistically significant increase in the number of micronucleated normochromatic erythrocytes in peripheral blood was detected by means of Giemsa staining 7 days after the last treatment. These results confirm the usefulness of the supravital acridine orange staining method to evaluate micronucleus induction in mouse peripheral blood.     

Cytogenetic mechanisms in the selective toxicity of cyclophosphamide analogs and metabolites towards avian embryonic B lymphocytes in vivo.

Cyclophosphamide (CP) is selectively toxic to avian and mammalian B lymphocytes, but the mechanisms of action are incompletely understood. We used a structure-activity approach to determine the cytogenetic mechanisms underlying the selective lymphoid toxicity in chicken embryos at 18-19 days of incubation. Two doses of 5-bromo-2'-deoxyuridine (BrdU; 3 mg/200 microliters x 2) were pipetted onto the inner shell membrane to label lymphocyte DNA over 20 h. A single dose of the CP analogs or metabolites was given 1 h after the initial BrdU application. After a terminal 3-h exposure to demecolcine to block cells in metaphase, the embryos were sacrificed at hour 20, and their bursae and thymi were removed for cytogenetic processing. Microscope slide preparations of metaphases were stained by the fluorescence-plus-Giemsa technique to differentiate the sister chromatids for an assessment of sister-chromatid exchange (SCE) induction and cell cycle progression based on replication cycle-specific staining patterns. Isophosphamide (1.25-40 mg/kg), phosphoramide mustard (0.7-45.7 mg/kg), and 4-methylcyclophosphamide (1.3-42.1 mg/kg) selectively damaged B cells as shown by dose-related reductions in the mitotic activity, inhibition of cell cycle kinetics, and approximately 9-15-fold increases in the SCE frequency above control. B cells were up to 392 times more susceptible to the toxicity of these three bifunctional alkylating agents compared to T cells based on reductions in the mitotic activity. At most of the drug doses tested, the T-cell mitotic index was not depressed significantly and was usually higher than the control value by as much as 50-60%. Importantly, monochloroethylcyclophosphamide (70-245 mg/kg; monofunctional alkylation) did not induce differential lymphoid toxicity, although a 9-fold increase in the SCE frequency of B cells was observed at the highest dose. Didechlorocyclophosphamide (181-422 mg/kg; acrolein generation only) was a weak SCE inducer (approximately 1.8-fold increase) and was not selectively toxic to B cells. Our data show that selective toxicity to B lymphocytes is strongly associated with bifunctional alkylation via the chloroethyl groups rather than with monofunctional alkylation and acrolein-mediated damage. In addition, the results with phosphoramide mustard and 4-methylcyclophosphamide emphasize that aldehyde dehydrogenase activity is not the primary determinant in the relative sparing of T lymphocytes in vivo.     

Effect of Acetaminophen Alone and in Combination with Morphine and Tramadol on the Minimum Alveolar Concentration of Isoflurane in Rats

Background

It has been observed that acetaminophen potentiates the analgesic effect of morphine and tramadol in postoperative pain management. Its capacity as an analgesic drug or in combinations thereof to reduce the minimum alveolar concentration (MAC) of inhalational anesthetics represents an objective measure of this effect during general anesthesia. In this study, the effect of acetaminophen with and without morphine or tramadol was evaluated on the isoflurane MAC.

Methods

Forty-eight male Wistar rats were anesthetized with isoflurane in oxygen. MAC_ISO was determined from alveolar gas samples at the time of tail clamping without the drug, after administering acetaminophen (300 mg/kg), morphine (3 mg/kg), tramadol (10 mg/kg), acetaminophen (300 mg/kg) + morphine (3 mg/kg), and acetaminophen (300 mg/kg) + tramadol (10 mg/kg).

Results

The control and acetaminophen groups did not present statistically significant differences (p = 0.98). The values determined for MAC_ISO after treatment with acetaminophen + morphine, acetaminophen + tramadol, morphine, and tramadol were 0.98% ± 0.04%, 0.99% ± 0.009%, 0.97% ± 0.02%, and 0.99% ± 0.01%, respectively.

Conclusions

The administration of acetaminophen did not reduce the MAC of isoflurane and did not potentiate the reduction in MAC_ISO by morphine and tramadol in rats, and therefore does not present a sparing effect of morphine or tramadol in rats anesthetized with isoflurane.     

The absorption and metabolism in rats of small oral doses of dimethylnitrosamine. Implication for the possible hazard of dimethylnitrosamine in human food.

1. Groups of rats were given one dose of the carcinogen dimethylnitrosamine by gastric intubation. The dose was varied between 10mg/kg body wt. and 1 microgram/kg body wt. 2. The dose was rapidly absorbed. 3. The methylation of liver DNA resulting from the administration of this carcinogen was proportional to dose. This suggests that small doses are absorbed from the gut with no more loss than large doses. 4. As the dose was decreased there was a disproportionately greater decrease in the alkylation of kidney DNA, and when the dose was less than 40 microgram/kg body wt. the methylation of kidney DNA was no longer detectable. This possibly explains why small amounts of dimethylnitrosamine in the diet do not induce kidney tumours. 5. Comparison of the relative alkylation of liver DNA and kidney DNA resulting from an oral and from an intravenous dose of dimethylnitrosamine suggest that small amounts of dimethylnitrosamine absorbed into the portal blood from the gut are completely metabolized by the liver and do not enter the general circulation. 6. The implications of these results for the possible hazard of dimethylnitrosamine in human food is discussed.     

The nNOS inhibitor,AR-R17477AR,prevents the loss of NF68 immunoreactivity induced by methamphetamine in the mouse striatum

The present study examined the time-course and regionally-selective changes in the levels of the neurofilament protein NF68 in the mouse brain induced by methamphetamine (METH). The ability of low ambient temperature, or of the specific neuronal nitric oxide synthase (nNOS) inhibitor AR-R17477AR, to protect against both long-term striatal NF68 and dopamine loss induced by METH (3 mg/kg, i.p.) was also studied. Seven days after METH administration (3, 6 and 9 mg/kg, i.p., three times at 3 h intervals), mice showed a reduction of about 40% in immunoreactivity for NF68 in the striatum. This effect was not produced in cortex after METH administration at the dose of 3 mg/kg. No difference from controls was observed when measurements were carried out 1 h and 24 h after the last METH injection at the dose of 3 mg/kg. The loss of NF68 immunoreactivity seems to be associated with the long-term dopamine depletion induced by METH, since no change in serotonin concentration is observed in either the striatum or cortex 7 days after dosing. Animals kept at a room temperature of 4 degrees C showed a loss of NF68 similar to those treated at 22 degrees C but an attenuation of dopamine depletion in the striatum. Pre-treatment with AR-R17477AR (5 mg/kg, s.c.) 30 min before each of the three METH (3 mg/kg, i.p.) injections provided complete protection against METH-induced loss of NF68 immunoreactivity and attenuated the decrease in striatal dopamine and HVA concentrations by about 50%. These data indicate that both the reduction of NF68 immunoreactivity and the loss of dopamine concentration are due to an oxidative stress process mediated by reactive nitrogen species, and are not due to changes in body temperature.     

The specificity of different classes of ethylating agents toward various sites in RNA.

The alkyl products of neutral in vitro ethylation of TMV-RNA by [14C]diethyl sulfate, [14C]ethyl methanesulfonate, and [14C]ethylnitrosourea have been determined and found to differ significantly depending on the ethylating agent. Diethyl sulfate and ethyl methanesulfonate ethylate the bases of TMV-RNA in the following order: 7-ethylguanine greater than 1-ethyladenine, 3-ethylcytidine greater than 7-ethyladenine, 3-ethyladenine, O6-ethylguanosine, 3-ethylguanine. Ethyl methanesulfonate was more specific for the 7 position of guanine, and other derivatives were found in lesser amounts than with diethyl sulfate. Neither reagent caused the formation of detectable amounts (smaller than 0.26 percent) of 1-ethylguanine, 1,7-diethylguanine, N2-ethylguanine, N6-ethyladenine, N4-ethylcytidine, or 3-ethyluridine. Identified ethyl bases account for over 85% of the total radioactivity of [14C]ethyl methanesulfonate and [14C]diethyl sulfate treated TMV-RNA. Phosphate alkylation accounts for about 13 and 1%, respectively, In contrast, [14C]ethylnitrosourea-treated TMV-RNA, while reacting to a similar extent (15-70 ethyl groups/6400 nucleotides), is found to cause considerably more phosphate alkylation. Upon either U4A RNase or acid hydrolysis up to 60% of the radioactivity is found as volatile ethyl groupw in the form of [14C]ethanol, and a further 15% appears to be primarily ethyl phosphate and nucleosides with ethylated phosphate. Of the remaining radioactivity, half is found as O6-ethylguanosine, the major identified ethyl nucleoside. Other ethyl bases found in ethylnitrosourea-treated TMV-RNA are 7-ethylguanine greater than 1-ethyladenine, 3-ethyladenine, 7-ethyladenine, 3-ethylcytidine, and 3-ethylguanine. It appears that ethylnitrosourea preferentially alkylates oxygens, and that formation of phosphotriesters is by far the predominant chemical event. Since the number of ethyl groups introduced into TMV-RNA by ethylnitrosourea is similar to the number of lethal events, one may conclude that phosphate alkylation leads to loss of infectivity. None of the three ethylating agents studied are strongly mutagenic on TMV-RNA or TMV. The role of phosphate alkylation in regard to in vivo mutagenesis and oncogenesis remains to be established. At present it appears possible that the extent of this reaction may correlate better with the oncogenic effectiveness of different ethylating agents, than the extent of any base reaction. Unfractionated HeLa cell RNA is ethylated primarily in acid labile manner even by diethyl sulfate and ethyl methanesulfonate, a fact that is attributed to its high content of low molecular weight trna rich in terminal phosphates which alkylate readily.