Effect of disulfiram on the alkylation of rat liver DNA by nitrosodiethylamine

Summary The extent of ethylation of guanine in 7 N and O 6 position in rat liver DNA was studied after chronic feeding with unlabelled nitrosodiethylamine followed by a single application of the ¹⁴C labelled drug. Disulfiram inhibits the alkylation of rat liver DNA. It was also shown that a discontinuous dosage of disulfiram protects against the alkylating effect of ¹⁴C nitrosodiethylamine only for a distinct period of time.

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Abbreviations DSF Disulfiram - NDEA Nitrosodiethylamine - DNMA Nitrosodimethylamine     

Influence of a prolonged treatment with disulfiram andd(-)penicillamine on nitrosodiethylamine-induced biological and biochemical effects in rats

Summary The influence of a prolonged treatment with disulfiram (DSF) andd(-)penicillamine (PA) on biological and biochemical effects induced by nitrosodiethylamine (NDEA) was studied in rats. The combination of NDEA and DSF led to a massive and early development of esophageal tumors, which were fatal to the animals. No liver tumors were observed in this group, whereas PA in combination with NDEA led to an increased development of liver tumors compared with NDEA alone. In the last two groups, only incidental tumors of the esophagus were observed. Nasal cavity tumors also appeared earlier in the animals treated with DSF and NDEA than in animals treated with NDEA alone or with NDEA plus PA. At a biochemical level, DSF led to a significant inhibition of hepatic anilinehydroxylase and nitroso-dimethylaminedemethylase in contrast to PA, which had no influence on these enzymes. The reduced activities of these drug-metabolizing enzymes did not appear to be related to gross cytochrome P450 content. Highly significant increases in glutathione content and glutathione-S-transferase activity (GSH/GST) were induced by DSF but not by PA. Because N-nitrosodiethylamine requires enzymatic activation to form the ultimate carcinogen, it is suggested that the observed inhibition of nitrosamine-transforming enzymes in the liver during DSF treatment leads to an increased amount of intact nitrosamines in other organs, e.g., in the esophagus, where it could be transformed to the ultimate carcinogen. DSF treatment alone or in combination with NDEA leads to an accumulation of trace elements in the liver, whereas PA eliminated copper and cobalt. The possible influence of these elements on tumor development is discussed in part II of this study.Abbreviations AH anilinehydroxylase - cyt P450 cytochrome P450 - cyt b5 cytochrome b5 - DDTC diethyldithiocarbamic acid - DSF disulfiram - NDEA nitrosodiethylamine - NDMA nitrosodimethylamine - NDMA-dem nitrosodimethylamine-demethylase - PA d(-)-penicillamine - GSH glutathione, reduced form - GST glutathione-S-transferase - NADP nicotinamide-adenine dinucleotide phosphate - NADH nicotinamide-adenine dinucleotide, reduced - TCA trichloroacetic acid Preliminary results of this study were presented at the 8th Drug Metabolism Workshop in Liège, Belgium, September 1982     

Influence of a prolonged treatment with disulfiram andd-penicillamine on nitrosodiethylamine-induced biological and biochemical effects

The influence of a 28-week treatment with disulfiram (DSF), D-penicillamine (PA), and nitrosodiethylamine (NDEA), as well as with a combination of DSF or PA with NDEA on the concentrations of eight essential trace elements in the whole liver tissue of rats was measured by means of neutron activation analysis. While NDEA treatment lowered the Zn content of the liver, DSF alone or in combination with NDEA enhanced the Zn and Se concentration by 50%-80%. Co, Cu, and Cd levels were increased by factors of 10, 60, and 110, respectively. The Mo concentration was decreased by 50% after DSF administration. PA reduced Cu, Co, and Zn in the liver. PA/NDEA treatment also lowered Cu, Co, and Zn content, but there was no strengthening effect of PA on the decrease in Zn observed with NDEA. The change of trace element concentrations, especially of Cu, is discussed with regard to the observed tumor induction in the liver, which tended to be increased by a combined NDEA/PA administration compared with NDEA treatment alone, whereas a protective action of DSF against NDEA induced liver tumors could not be established.     

Comparative studies on hepatic DNA alkylation in rats by N-nitrosomethylethylamine and N-nitrosodimethylamine plus N-nitrosodiethylamine

Summary The purpose of this study was to determine whether quantitative differences in hepatic DNA methylation or ethylation are sufficient to explain differences in the carcinogenicity of N-nitrosomethylethylamine (NMEA), N-nitrosodimethylamine (NDMA), and N-nitrosodiethylamine (NDEA). Methylation and ethylation of hepatic DNA were determined in male Fischer 344 rats following a single IP dose of NMEA, NDMA, NDEA or an equimolar mixture of NDMA plus NDEA. The total nitrosamine dose ranged from 0.05 to 0.25 mmol/kg. After 5 h survival, hepatic DNA was extracted by adsorption onto hydroxyapatite. Acid hydrolysates were analyzed by cation exchange HPLC with fluorescence detection. DNA methylation by NMEA (170 mol O⁶-methylguanine/mol guanine at 0.1 mmol/kg) was comparable to that observed in animals given an equimolar mixture of NDMA plus NDEA, indicating that NMEA is one-half as effective a methylating agent as NDMA. In contrast, the amount of ethylation by NMEA (5.9 mol O⁶-methylguanine/mol guanine at 0.1 mmol/kg) was approximately 4 times less than that observed in animals treated with an equimolar mixture of NDMA and NDEA. The presence of NDEA had little effect on DNA methylation by NDMA, suggesting that neither synergism nor competition occurs in the simultaneous activation of these two nitrosamines. The role of -hydroxylation of NMEA as a metabolic pathway that reduces the extent of DNA ethylation is discussed.Presented at the SEK workshop DNA Adducts and Chemical Carcinogenesis, Tübingen, February 28–March 1, 1986     

Proline dithiocarbamate inhibitsN-nitrosodiethylamine induced liver carcinogenesis

Summary A study was conducted to determine the toxicity of different dithiocarbamates and of disulfiram. In an experiment showing the cytotoxicity against murine spleen lymphocytes, proline dithiocarbamate (PDTC) and thioproline dithiocarbamate showed the lowest toxicity. Therefore one of them was selected and different doses of the hydrophilic PDTC were checked for their ability to affect the development of liver and oesophagus tumours induced in BD-6 rats byN-nitrosodiethylamine (NDEA). Rats were injected i.p. with 80 mg/kg NDEA once weekly for 10 weeks. Administration of PDTC, 1 h before and 24 h after the carcinogen, markedly decreased the number of rats developing NDEA-induced hepatocellular carcinoma and liver haemangioendothelioma. A 59%–77% reduction in the incidence of liver tumours was found in the different groups when the carcinogen was administered in combination with the inhibitor. For least 40 weeks after the start of the experiment PDTC protected the liver from NDEA carcinogenesis and did not shift the tumour development to any other organ. PDTC did not significantly affect the weight gain of the experimental animals. We conclude that parenteral administration of PDTC seems to represent a promising approach in chemoprevention of liver carcinogenesis.Abbreviations NDEA N-nitrosodiethylamine - PDTC proline dithiocarbamate - SDTC sarcosine dithiocarbamate - TDTC throproline dithiocarbamate - DDTC diethyldithiocarbamate     

Effects of potassium ethylxanthogenate on nitrosodiethylamine-induced DNA damage and on liver carcinogenesis

BD-6 rats were injected with 80 mg/kg N-nitroso-diethylamine weekly for 10 weeks. Addition of 270 mg/kg potassium ethylxanthogenate weekly reduced significantly the number of rats developing NDEA-induced malignant liver tumors. Ethylxanthogenate decreased the total number of liver tumors induced by the carcinogen to 6 as compared to a total of 29 neoplasms in animals treated only with NDEA. In acute experiments potassium ethylxanthogenate markedly decreased the exhalation of 14CO2 derived from 14C-NDEA. The amount of the nonmetabolized carcinogen increased in the urine of ethylxanthogenate protected rats only 4% of the given dose. Initial DNA damage, single strand breaks and alkali-labile sites, was determined by alkaline sucrose gradients and in protected animals was minimal for at least 24 hours after NDEA administration. It appears that the production of less initial DNA damage may be important for the further course of liver carcinogenesis induced by relatively large doses of nitrosodiethylamine.     

Metabolism of methylazoxymethanol acetate in the F344 rat and strain-2 guinea pig and its inhibition by pyrazole and disulfiram

Summary The basic parameters of the metabolism of methylazoxymethanol-(N,N-methyl-¹⁴C) acetate, in terms of exhaled ¹⁴CO₂, urinary metabolites, and inhibition by pyrazole and disulfiram, were examined in F344 rats and strain-2 guinea pigs. After a 18.7 mg/kg SC dose, 45% (6 h) and 49.5% (24 h) was exhaled as ¹⁴CO₂, and 6.6% (24 h) of the radioactivity was excreted in the urine by the rats. After identical treatment, 36.5% (6 h) and 39.5% (24 h) was exhaled as ¹⁴CO₂ and 3.5% (24 h) was excreted in the urine by the guinea pigs. Urea-¹⁴C and methylazoxymethanol(-¹⁴C) were the major urinary metabolites. In both species, pretreatment with pyrazole (40 or 360 mg/kg, IP) caused a significant reduction of exhaled ¹⁴CO₂ and an increase in the amount of urinary methylazoxymethanol(-¹⁴C). Similar but less pronounced effects were observed after pretreatment of rats with disulfiram (1 g/kg, PO). These results are discussed with respect to possible enzyme systems involved in the metabolic activation of methylazoxymethanol in vivo.Abbreviations AM azomethane - AOM azoxymethane - DMH 1,2-dimethylhydrazine - HPLC high-performance liquid chromatography - MAM methylazoxymethanol - MAMOAc methylazoxymethanol acetate Dedicated to Professor Hermann Druckrey on the occasion of his 80th birthdaySupported, in part, by Grant CA-31012 from the National Cancer Institute     

Metabolism of,and DNA methylation by,N-nitrosomethylbenzylamine in chicken

Summary The metabolism of ¹⁴C-nitrosomethylbenzylamine (NMBA) was studied in chicken. Following a single IV dose of 2 mg/kg, ¹⁴C-NMBA was cleared from the blood with a half-life of 3.8 min. At 10 min after administration ¹⁴C-NMBA was totally metabolized in the liver, whereas in the esophagus no measurable metabolic degradation had taken place.Maximum exhalation of radioactive CO₂ occurred 1 h after IV administration of NMBA, and 11% of the total radioactivity had been exhaled as CO₂ by 8 h. These results are compared with data on the metabolism of NMBA in the rat.The analysis of methylated bases in the DNA of different organs of chicken revealed that 7-me guanine was formed in all organs. The highest amount of 0⁶-me guanine was found in liver DNA, followed by kidney DNA. O⁶-me guanine was not detectable in any other organ. The O⁶-/7-me guanine ratio in DNA was calculated to be 0.05 and 0.02 for liver and 0.01 for kidneys.Abbreviations O⁶-meG O⁶ methylguanine - 7-meG 7-N-methylguanine - G guanine - A adenine - NMBA N-nitrosomethylbenzylamine Dedicated to Prof. Dr. H. Druckrey on the occasion of his 80th birthdayRecipients of an Alexander von Humboldt-Fellowship     

ω-3鱼油脂肪乳剂对重症急性胰腺炎患者炎症免疫与器官功能的影响

  目的 探讨静脉应用ω-3鱼油脂肪乳剂对重症急性胰腺炎（SAP）患者炎症反应、免疫功能及器官功能的影响。方法 临床确诊为SAP患者随机分为常规治疗组(A组)和常规+鱼油治疗组(B组),A组常规治疗（抗感染、抑酶、胃肠减压、通便导泻及器官功能支持等）;B组：按A组的常规治疗后静脉滴注10%ω-3鱼油脂肪乳剂0.2 g·kg^-1·d^-1,连续应用14 d。观察治疗前、治疗后7天、治疗后14天C反应蛋白（CRP）、TG、TC、T细胞亚群（CD⁺₄、CD⁺₈）水平及补体C₃、补体C₄水平;测腹内压（膀胱压）,记录急性生理与慢性健康评估Ⅱ（APACHEⅡ）、达到液体负平衡时间、肠内营养时间、住ICU时间及28 d病死率。结果 53例SAP患者入选本研究,其中6例因肾衰竭行床旁血液净化治疗,2例自行退出,最后45例纳入本研究。B组（22例）患者治疗后14天CD⁺₄ T细胞、CD⁺₄/CD⁺₈、补体C₃改善明显优于A组（23例）;B组治疗后7天、14天CRP、腹内压、APACHEⅡ评分较A组下降更明显;B组患者开始液体负平衡时间［(3.55±0.86) d］明显短于A组［（4.61±1.12）d］,开始肠内营养时间［（3.86±1.17）d］明显早于A组［（5.30±1.61）d］,住ICU时间［(11.36±3.13)d比（14.96±4.0）d］、28 d病死率（4.5%比4.4%）2组差异无统计学意义（P>0.05）。结论 SAP患者静脉应用鱼油脂肪乳剂可减轻炎性反应,改善免疫功能,并可早期达到液体负平衡管理及恢复肠道功能,为SAP提供新的、有效的治疗手段。     

Coadministration of disulfiram and lorazepam in the treatment of alcohol dependence and co-occurring anxiety disorder: an open-label pilot study

ABSTRACT

Background: Anxiety is common among persons with alcohol use disorder during early abstinence from alcohol. Although benzodiazepines are effective for short-term treatment of anxiety, they are rarely used beyond acute detoxification due to concerns about misuse or interactions with alcohol. Objectives: We conducted an open-label trial to explore the effects of coadministering lorazepam and disulfiram to alcohol-dependent patients with anxiety disorder symptoms. The rationale for this model is to minimize the risks of the benzodiazepine, while also potentially enhancing adherence to disulfiram. Methods: Forty-one participants with DSM-IV alcohol dependence who also met syndromal criteria for anxiety disorder with or without co-occurring major depressive syndrome initiated treatment with lorazepam (starting dose 0.5 mg three times daily) and disulfiram (starting dose 500 mg three times weekly). Participants received 16 weeks of monitored pharmacotherapy with manualized medical management. Results: Adherence to treatment decreased steadily with time (85.4% at 4 weeks, 36.6% at 16 weeks). Participants showed significant increases in percent abstinent days during treatment and at 24 weeks follow-up. Large reductions in anxiety, depression, and craving were observed during treatment, and improvement remained significant at 24 weeks. Duration of adherence with disulfiram strongly predicted abstinence at 16 weeks. There was no evidence of misuse of lorazepam or dose escalation during the study. Conclusion: Lorazepam can be safely used for short-term treatment of anxiety in combination with disulfiram treatment of alcohol use disorder. However, it is not clear that making lorazepam dispensing contingent on adherence to disulfiram enhances retention in disulfiram treatment.     

Evaluation of chemopreventive effect of Fumaria indica against N-nitrosodiethylamine and CCl4-induced hepatocellular carcinoma in Wistar rats

ObjectiveTo investigation the chemopreventive potential of Fumaria indica (F. indica) extract (FIE) on N-nitrosodiethylamine and CCl₄-induced hepatocarcinogenesis in Wistar rats.MethodsThe experimental animals were divided into six groups (n=6). Hepatocellular carcinoma was induced by single intraperitoneal injection of N-nitrosodiethylamine (NDEA) in normal saline at a dose of 200 mg/kg body weight followed by weekly subcutaneous injections of CCl₄(3 mL/kg/week) for 6 weeks, as the promoter of carcinogenic effect. After administration of the carcinogen, 200 and 400 mg/kg of FIE were administered orally once a day throughout the study. At the end of 20 weeks, the body weight, liver weight and relative liver weight were measured. The percentage of nodule incidence and liver cancer markers such as aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP), γ-glutamyl transferase (γ-GT), total bilirubin level (TBL), α-feto protein (AFP) and carcinoembryonic antigen were estimated along with histopathological investigation in experimental groups of rats.ResultsObtained results demonstrated that the cotreatment with FIE significantly prevented the decrease of the body weight and also increased in relative liver weight caused by NDEA. The treatment with FIE significantly reduced the nodule incidence and nodule multiplicity in the rats after NDEA administration. The levels of liver cancer markers such as AST, ALT, ALP, γ-glutamyl transferase, TBL, AFP and carcinoembryonic antigen were substantially increased by NDEA treatment. However, FIE treatment significantly reduced the liver injury and restored the entire liver cancer markers. Histological observations of liver tissues too correlated with the biochemical observations.ConclusionsThese finding powerfully supports that F. indica exert chemopreventive effect by suppressing the tumor burden and restoring the activities of hepatic cancer marker enzymes on NDEA and CCl₄-induced hepatocarcinogenesis in Wistar rats.     

Modifying effects of disulfiram on DNA adduct formation and persistence of benzaldehyde in N-nitroso-N-methyl-benzylamine-induced carcinogenesis in rats

Summary Whereas disulfiram (DSF) is known to inhibit tumor formation resulting from a number of chemical carcinogens, such inhibition does not apply to nitrosamines. In the present study, biochemical and morphological findings were examined to elucidate the effect of DSF on long-term application of N-nitroso-N-methylbenzylamine (NMBA). HPLC and fluorescence detection were used to determine O⁶-methylguanine (O⁶-MG) in DNA obtained from the respiratory tract of rats subjected to long-term simultaneous application of DSF and NMBA. After 2 days of treatment, more O⁶-MG was detected in the proximal portion of the respiratory tract, including the trachea and main bronchi, than in the distal portion. The findings were reversed after 10 and 30 days, at which time formation of the DNA adduct was substantially higher in the distal portion of the respiratory tract, despite increases in both portions. The biochemical results corresponded to morphological findings. Initially, mereased numbers of metabolizing goblet cells appeared in mucous cell hyperplasia in the proximal respiratory tract. Subsequently, the hyperplasia migrated to distal regions of the respiratory tract; at this stage, the goblet cells disappeared from the proximal portion, which now revealed toxic degeneration, atrophy and subsequent squamous metaplasia of the mucous lining and squamous papillomas.At various times during a 40-day period, 2 to 7 times more O⁶-MG in pulmonary DNA was detected in rats treated with DSF and NMBA, than with NMBA alone, whereby distinct amounts of O⁶-MG were found in the latter animals. In contrast to the above-mentioned morphological findings, no morphological alterations occurred in the respiratory tract of the animals treated with NMBA alone. It is therefore conceivable that the above pathological lesions resulted not merely from the presence of DNA adducts, but also from an additional, previously unspecified effect.As benzaldehyde (BA) is formed in equimolar amounts in NMBA metabolism and DSF has been demonstrated to inhibit aldehyde metabolism, this aldehyde is a possible candidate for such an effect. In the present study, rats were therefore treated with BA, DSF, or NMBA, or combinations thereof. Histomorphological evaluation of these experiments revealed that long-term application of BA alone led to the following alterations in the respiratory tract: goblet cell hyperplasia, hyperplasia of the peribronchial lymphatic system, mucous epithelial atrophy and accompanying peerivasculitis — the same alterations seen under long-term application of NMBA and DSF. Furthermore, these changes were most pronounced in the group with concomitant application of NMBA, DSF, and BA. It is therefore conceivable that BA plays a role in pathological changes observed under the influence of NMBA.Presented at the SEK workshop DNA Adducts and Chemical Carcinogenesis, Tübingen, February 28–March 1, 1986Supported by Breuninger Stiftung, D-7000 Stuttgart; AWG-Analysen-Geräte GmbH, D-7970 Leutkirch, FRG; Asta-Werke AG, D-6000 Frankfurt am Main. FRG; Cyanamid GmbH, D-8190 Wolfratshausen, FRG, Farmitalia Carlo Erba GmbH, D-7800 Freiburg i Br., FRG     

Interleukin-6 induces proliferation of rat hepatocytes in vivo

Background/Aims: The aim of this study was to assess the effect of interleukin-6 (IL-6) on the proliferation of hepatocytes and to study the interaction between IL-6 and hepatocyte growth factor (HGF) in vivo.Methods: IL-6 was injected at a dose of 200 μg/mg subcutaneously into rats every day for 14 days. Liver and blood samples were obtained at 1, 3, 7 and 14 days during IL-6 administration. Hepatocyte proliferative activity of sera was measured using ³H-thymidine incorporation into cultured rat hepatocytes. To evaluate the proliferative activity of the hepatocytes in tissue sections, hepatic DNA content and immunostaining of the liver tissue sections for proliferating cell nuclear antigen (PCNA) were performed. Plasma HGF levels were measured using specific EIA. In addition, total RNA was extracted from the liver and expression of HGF mRNA was detected by RT-PCR.Results: The DNA contents of liver taken from IL-6-treated rats were increased during IL-6 administration compared with untreated rats. Sera taken from IL-6-treated rats at various intervals during administration also significantly increased ³H-thymidine incorporation by cultured rat hepatocytes compared with sera from untreated rats, suppressing ³H-thymidine incorporation at day 1 and 3 by anti-HGF antibody. IL-6 itself did not increase ³H-thymidine incorporation. Increased expression of PCNA in these hepatocytes was noted from 1 day after IL-6 administration, and at 14 days, the number of PCNA-positive cells was sevenfold greater than in the livers of untreated rats. However, plasma HGF levels showed a peak at day 1, decreased gradually from day 3, and became undetectable by day 14. HGF mRNA expression in livers of IL-6-treated rats was suppressed from day 3 to day 14 of IL-6 administration.Conclusions: These data show that IL-6 induces an early phase of liver cell growth in vivo and suggest that an increase level of HGF mediates this effect.     

Role of prostaglandins in the early phases of experimental gastric carcinogenesis in the rat

Summary The study was initiated to evaluate the effect of N-methyl-N-nitro-N-nitrosoguanidine (NG) on gastric intraluminal prostaglandin release during a 30-day treatment period and to investigate the effect of a stable prostaglandin E¹ analogue (misoprostol) on NG-induced gastric mucosal damage during the same time period. Samples of gastric juice (1 h) were obtained from 40 male Sprague-Dawley rats with chronic gastric fistulas, in basal conditions and after 5, 15 and 30 days of continuous oral administration of NG (120 mg/l) or tap water. Aliquots of gastric juice were titrated with 0.1 M NaOH. Other aliquots were extracted with ethyl acetate and subjected to specific radioimmunoassay for prostaglandin E₂. The severity of gastric mucosal lesions was evaluated in 60 rats after 5 days and 30 days of continuous oral administration of NG (120 mg/l) or NG plus misoprostol (200 g/kg^-1/day^-1) or tap water, and a histological study was carried out. Administration of NG induced a significant decrease of gastric intraluminal prostaglandin E₂ concentration at 15 and 30 days. Oral administration of misoprostol, at non-antisecretory doses, protected the rats against NG-induced gastric mucosal damage. Prostaglandins may be involved in the early phases of experimental gastric carcinogenesis.This work was supported by grants from the Ministry of Education (MPI)     

O6-methylguanine repair in liver cells in vivo: Comparison between G1- and S-phase of the cell cycle

Summary To compare the formation and persistance of alkylated DNA bases in the G₁- and S-phase compartments in liver in vivo, regnerating rat liver was exposed to [¹⁴C]dimethylnitrosamine (0.57 mg/kg, IP injection) or N-[methyl ¹⁴C]-N-nitrosourea (3.3 mg/kg, intraportal injection) during the G₁ phase of the cell cyle (12 h after partial hepatectomy), or at 24 h after partial hepatectomy with 30% hepatocytes in DNA synthesis, or at 43 h after partial hepatectomy, 4 h after an hydroxyurea block from 14 to 39 h after operation with 80% hepatocytes in DNA synthesis. At 120 min after dimethylnitrosamine and 90 s, 5, 10, or 60 min after the intraportal pulse of N-methyl-N-nitrosourea the molar fractions of 7-methylguanine (7megua), O⁶-methylguanine (O⁶megua), and 3-methyladenine (3mead) and of metabolically labeled guanine were determined from DNA hydrolysates by Sephadex-G10 radiochromatography. After dimethylnitrosamine only minor differences were observed for 7megua formation in the three groups; the 3mead/7megua ratio remained constant irrespective of the number of cells in S phase. In contrast, the O⁶megua/7megua ratio revealed a loss of O⁶megua, the extent of which appeared proportional to the fraction of DNA-synthesizing cells in the liver. The rapid loss of O⁶megua in S-phase cells was confirmed after intraportal administration of N-methyl-N-nitrosourea. During the first 10 min after the methylnitrosourea pulse the O⁶megua/7megua ratio was constant in G₁ cells and dropped from 90 s to 10 min by about 15% in liver containing 30% S-phase cells and by about 40% with 80% cells in DNA synthesis. DNA-synthesizing hepatocytes are apparently endowed with a higher O⁶megua DNA transferase activity than nonproliferating liver cells. The rapid, though exhaustible elimination of O⁶megua during S-phase might result in partial protection of DNA-synthesizing cells from base-mispairing and/or from hypomethylation at G-C sites.Dedicated to Professor Hermann Druckrey on the occasion of his 80th birthdaySupported by grants from the Deutsche Forschungsgemeinschaft and the Dr. Mildred Scheel-Stiftung     

Expression of Tumor Necrosis Factor-α and Interleukin-6 Cell-Surface Receptors of the Alveolar Macrophage in Alcohol-Treated Rats

The hypothesis was tested that alcohol may modulate alveolar macrophage cytokine receptors, thus interfering in lung immune defense mechanisms. Male rats were treated with alcohol either acutely (7 hr continuous intravenous alcohol infusion at a rate of 30 mg/100 g body weight/hr after a priming dose of 175 mg/100 g body weight) or chronically (feeding an alcohol-containing liquid diet for 12–14 weeks). Three hr before killing, the rats received an intravenous injection of Gram-negative bacterial lipopolysaccharide (LPS; Escherichia coll, O26:B6, 100 μg/100 g body weight). After anesthesia with sodium pentobarbital, the trachea was cannulated, and the lungs excised and lavaged to obtain alveolar macrophages. The recovered cells were used to measure the binding of recombinant human [¹²⁵I]tumor necrosis factor-α (TNF-α) and [¹²⁵I]interleukin-6 (IL-6). K_d and B_max, were determined at 4°C, thus reflecting only the cell-surface binding sites and their affinity. Two binding sites were detected for both cytokines: high-affinity (K_d1 in the range of 20–110 pM), low-capacity (B_max, in the range of 1–13 fmol/10⁵ cells), and low-affinity (K_d2 in the range of 0.6–1.3 nM), high-capacity (B_max2 in the range of 34–100 fmol/10⁵ cells). Acute alcohol treatment significantly decreased B_max1 (39%) and B_max (79%) for TNF-α, whereas chronic alcohol feeding abrogated the B_max1 (B_max1= 0), without affecting B_max2. In the acute group, LPS had an effect similar to that of alcohol. Alcohol administration did not modify the LPS effects. The following changes were monitored for IL-6 binding. Acute alcohol treatment markedly reduced (86%) B_max2. LPS administration to saline-infused group increased K_d1 (122%) and B_max1 (197%), and significantly diminished (72%) B_max2 These effects of LPS were not altered by alcohol administration, with the exception of K_d1 which was decreased (43%) by LPS administration to acutely, alcohol-intoxicated rats. In the chronic treatment group, alcohol feeding increased B_max2 (426%), an effect that was not modified by LPS injection. These data suggest that: (1) both acute and chronic alcohol administration to rats differentially affects the alveolar macrophage cell-surface receptors for TNF-α and IL-6, and (2) alcohol-induced alterations in these two cytokine receptors may in part mediate alcohol effects on lung immune defense.     

Proline impairs energy metabolism in cerebral cortex of young rats

In the present study we investigated the effect of acute hyperprolinemia on some parameters of energy metabolism, including the activities of succinate dehydrogenase and cytocrome c oxidase and ¹⁴CO₂ production from glucose and acetate in cerebral cortex of young rats. Lipid peroxidation determined by the levels of thiobarbituric acid-reactive substances, as well as the influence of the antioxidants α-tocopherol plus ascorbic acid on the effects elicited by Pro on enzyme activities and on the lipid peroxidation were also evaluated. Wistar rats of 12 and 29 days of life received one subcutaneous injection of saline or proline (12.8 or 18.2 μmol/g body weight, respectively) and were sacrificed 1 h later. In another set of experiments, 5- and 22-day-old rats were pretreated for a week with daily intraperitoneal administration of α-tocopherol (40 mg/kg) plus ascorbic acid (100 mg/kg) or saline. Twelve hours after the last injection, rats received one injection of proline or saline and were sacrificed 1 h later. Results showed that acute administration of proline significantly reduced cytochrome c oxidase activity and increased succinate dehydrogenase activity and ¹⁴CO₂ production in cerebral cortex, suggesting that Pro might disrupt energy metabolism in brain of young rats. In addition, proline administration increased the thiobarbituric acid-reactive substances levels, which were prevented by antioxidants. These findings suggest that mitochondrial dysfunction and oxidative stress may be important contributors to the neurological dysfunction observed in some hyperprolinemic patients and that treatment with antioxidants may be beneficial in this pathology.     

Cell Proliferation and DNA Dependent DNA Polymerase Estimation in Acute Lymphoblastic Leukaemia during Treatment with Prednisone and Vincristine

The presence of DNA polymerase and primer-template DNA in lymphoblast nuclei by measuring the in vitro incorporation of ³H-thymidine-5′-triphosphate (³H-TTP) was studied in 10 patients with acute lymphoblastic leukaemia. Protein synthesis and various other cytokinetic parameters were also studied. After prednisone (P) administration a marked decrease in ³H-TTP labelling index (³H-TTP LI) was apparent together with an inhibition of ³H-leucine incorporation (³H-LEU LI) into lymphoblasts. A moderate decrease in ³H-TDR labelling index (³H-TDR LI) and a later decrease in mitotic index (MI) were seen. Single cell DNA measurements showed a depletion of ³H-TDR labelled lymphoblasts in early part of S-phase apparent at 24 h lasting up to 54 h after P administration. Vincristine given as a flash injection later in the study period caused an immediate rise of the MI, at the same time the P induced decline in ³H-TTP LI, ³H-TDR LI and ³H-LEU LI were continued in most patients. P is thought to damage the cells both in and outside the cell cycle. In the cell cycle the effect of P is an arresting effect in G₁.     

Inhibition of diethylnitrosamine-induced strand breaks in liver DNA by disulfiram

Summary Disulfiram (DSF) delayed the appearance of diethylnitrosamine (DEN)-induced strand breaks in liver DNA of rats. The fragmentation of liver DNA produced by DEN was studied 4 and 24 h after administration of the carcinogen on alkaline sucrose gradients. A single dose of 500 mg/kg DSF given 1 h prior to carcinogen treatment delayed for at least 4 h the DEN —induced strand breaks in liver DNA. A single DSF pretreatment, however, did not protect against carcinogen-induced strand breaks when observed 24 h after DEN injection. It is possible that continuous administration of DSF might inhibit the DEN-produced damage of the genetic material.

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Hemmung von Diäthylnitrosamin-induzierten Strangbrüchen in Leber DNS durch Disulfiram

Zusammenfassung Disulfiram (DSF) verzögert das Auftreten von Diäthylnitrosamin (DEN)-induzierten Strangbrüchen der DNS in Leberzellen von Ratten. Eine durch DEN erzeugte Fragmentierung der DNS wird 4 bzw. 24 Std nach der Carcinogengabe mit Hilfe eines alkalischen Sucrose-Gradienten untersucht. Eine Einzeldosis von 500 mg/kg DSF 1 Std vor dem Carcinogen verabreicht, verhindert Strangbrüche der Leber DNS für mindestens 4 Std. Die Vorbehandlung mit DSF schützt jedoch nicht vor Strangbrüchen nach einer längeren Periode von z. B. 24 Std. Möglicherweise verhindert eine fortgesetzte Behandlung mit DSF die DEN-induzierte Schädigung von genetischem Material.

     

Tumor Necrosis Factor-α Cell-Surface Receptors of Liver Parenchymal and Nonparenchymal Cells during Acute and Chronic Alcohol Administration to Rats

Tumor necrosis factor-α (TNF-α) has been shown to contribute to the alcohol [ethanol (ETOH)]-induced alteration of hepatic function. Therefore we tested the hypothesis that the hepatic action of TNF-α could be due, at least in part, to alterations in TNF-α cell-surface receptors of hepatic parenchymal (hepatocytes) and nonparenchymal (Kupffer and sinusoidal endothelial) cells. Rats were either acutely treated with ETOH by a primed, continuous 7-hr intravenous infusion of 20% (w/v) ETOH (30 mg/100 g body weight/h) or chronically fed an ETOH-containing liquid diet (5.2% ETOH, w/v, with ETOH as 36% of total calories) for 14 weeks. Control rats in the acute group were infused with sterile saline, whereas control rats in the chronic group were fed liquid diet containing dextrin to replace ETOH in isocaloric amounts. Three hr before killing, the rats were injected intravenously with Gram-negative bacterial lipopolysaccharide [(LPS) 100 /μg/100 g body weight] or saline. Hepatocytes, Kupffer cells, and sinusoidal endothelial cells were isolated after liver perfusion with collagenase (without pronase), separated by centrifugal elutriation, and used to determine the affinity (K_d) and capacity (B_max) of binding sites, using recombinant human-[¹²⁵l]TNF-α as the ligand. Two binding sites were detected on Kupffer cells and sinusoidal endothelial cells isolated from control animals: a high-affinity (K_d1, in the range of 150–200 PM), low-capacity (B_max1, in the range of 2–3 fmol/10⁶ cells) binding site and a low-affinity (K_d2, in the range of 2–9 nm), high-capacity (B_max2, in the range of 3–15 fmol/10⁶ cells) binding site. One binding site was detected on hepatocytes isolated from control rats (K_d1= 1.0 nm and a B_max= 95 fmol/10⁶ cells). Acute ETOH administration caused an increase in K_d1 on both Kupffer and sinusoidal endothelial cells; a decrease in K_d1 on hepatocytes; and an increase in K_d2, B_max1, and B_max2 on endothelial cells. A second binding site on hepatocytes (K_d2= 5.8 nm, S_max2= 186 fmol/10⁶ cells) was observed only in the ETOH-treated group after LPS administration. Chronic alcohol exposure markedly elevated K_d1 and S_max1 on hepatocytes. Overall, LPS-induced changes mimicked those induced by alcohol, except for a decrease in B_max1 on Kupffer cells of rats in the acute treatment group. Also, the liquid diet containing alcohol or dextrin abrogated the high-affinity, low-capacity binding sites on both Kupffer cells and sinusoidal endothelial cells, and low-affinity, high-capacity binding sites on the hepatocyte. After LPS administration, the high-affinity binding sites on Kupffer and sinusoidal endothelial cells were reexpressed. These data show that: (1) ETOH induces changes in both the capacity (B_max) and affinity (K_d) of TNF-α cell-surface receptors of hepatocytes, Kupffer cells, and sinusoidal endothelial cells; and (2) ETOH-induced changes are consistent with an increased sensitivity of these cells to the action of TNF-α.