Induction of tumours in rats by feeding nitrosatable amines together with sodium nitrite

Feed containing 0.2% allantoin or diphenhydramine (as the hydrochloride) or 0.1% chlorpheniramine (as the maleate), with or without 0.2% sodium nitrite, was given ad lib. to groups of 20 or 24 male and 20 or 24 female F344 rats for 106 wk. Groups of 24 male and 24 female F344 rats were given drinking-water that contained N,N-dimethyldodecylamine-N-oxide at a concentration of 0.1%, with or without 0.2% sodium nitrite, for 93 wk. Control rats were given untreated feed or drinking-water and nitrite-treated controls were given sodium nitrite at a concentration of 0.2% in feed or drinking-water. At the end of the treatment period the rats were given untreated feed and water and observed until death. There was little or no life-shortening effect in any treatment group. None of the four amines administered alone induced an increase in the incidence of any tumour in comparison with the untreated control groups. In the male rats given diphenhydramine, chlorpheniramine or N,N-dimethyldodecylamine-N-oxide concurrently with nitrite there was a significant increase in the incidence of liver neoplasms (hepatocellular carcinomas and neoplastic nodules). In the groups given untreated feed or drinking-water there were, respectively, five and three male rats that had liver tumours. In contrast the number of male rats with liver tumours was ten in the group given dimethyldodecylamine-N-oxide plus nitrite, 11 in that given diphenhydramine plus nitrite and 14 (eight with carcinomas) in the group given chlorpheniramine plus nitrite. These results suggest that the ingestion of dimethyldodecylamine-N-oxide, diphenhydramine hydrochloride or chlorpheniramine under conditions when they could be nitrosated with nitrite in the stomach might present an increased carcinogenic risk.     

Dose-response studies in carcinogenesis by nitroso-N-methyl-N-(2-phenyl)ethylamine in rats and the effects of deuterium substitution

A dose-response study of the carcinogenicity of nitroso-N-methyl-N-(2-phenyl)ethylamine was carried out in male Fischer 344 rats. The compound was given in drinking water at concentrations of 115, 28, 9·5, 3·2, 1·1 and 0·4 mg/litre. The highest concentration proved toxic leading to the early death of several animals; the remainder of this group were treated for 21 wk. All of the other concentrations were given for 33 wk, except the 28 mg/litre treatment which ceased at 30 wk. An additional group of rats was given 0·4 mg/litre for 104 wk. In all groups of animals, except those in the high-dose group that died early and those given 0·4 mg/litre for 33 wk, 50% or more of the animals had tumours of the oesophagus or forestomach or both when they died. In several groups the number of rats with these tumours approached 100%. The total dose of carcinogen received by the rats in the lowest dose group was 1·3 mg and 45% of them had tumours of the upper gastro-intestinal tract. The effect on carcinogenicity of labelling the nitrosamine with deuterium in either the methyl group or the α-methylene of the phenylethyl group was determined by treating groups of rats with equimolar concentrations of the deuterium-labelled and unlabelled nitrosamine. A very significant increase in carcinogenic effectiveness was observed with the compound containing deuterium in the α-methylene of the phenylethyl group, suggesting that methylation might not be the important event in carcinogenesis by this compound in rats.     

EGCG对实验性糖尿病大鼠肾脏的保护作用

目的观察表没食子儿茶素没食子酸酯(EGCG)对大鼠糖尿病肾脏的保护作用。方法采用链脲佐菌素(65 mg.kg-1)腹腔注射建立糖尿病大鼠模型,实验分4组:对照组、模型组、EGCG治疗组Ⅰ和治疗组Ⅱ,后两组于模型成功后1 wk和4 wk给予EGCG 5 mg.kg-1.d-1腹腔注射。分别测定各组4、8、12 wk时尿白蛋白/肌酐比值(A/C)。12 wk后,采用高效液相测定血浆同型半胱氨酸(tHcy)含量;观察肾脏损伤后病理形态学变化;应用免疫组化检测肾组织细胞外基质蛋白酶-9(MMP-9)的表达;生化分析肾脏氧化应激状态。结果模型组与对照组相比:大鼠肾重/体重明显升高;肾小球肥大,细胞增生,PAS阳性物质明显沉积;血浆tHcy含量及肾组织MMP-9的表达明显增加。经EGCG干预后大鼠生化指标和肾重/体重明显改善;肾脏的抗氧化能力增强和氧化应激降低;血浆tHcy含量下降和MMP-9的表达减弱。EGCG两治疗组相比较,其结果也存在差异。模型组大鼠尿A/C在不同时相点都维持在高水平,且随着时间的延长而逐渐升高,EGCG治疗组I在各时间点与模型组相比A/C明显降低(P<0.01),而治疗组Ⅱ在12 wk时明显低于模型组(P<0.05),在不同时间点治疗组I和治疗组Ⅱ之间存在差异。结论EGCG对糖尿病肾病具有保护作用,其作用机制可能是通过提高机体抗氧化应激能力,降低血浆tHcy含量,抑制肾组织MMP-9表达而减少糖尿病肾病损害。     

Liver medium-term bioassay in rats for screening of carcinogens and modifying factors in hepatocarcinogenesis.

The present report describes a study of the hepatocarcinogenic potential of a second large assay series of 94 compounds carried out using the rapid bioassay system (DEN-PH model) developed in this laboratory and based on the two-step concept of hepatocarcinogenesis. Male F344 rats were initially given a single dose of diethylnitrosamine (DEN, 200 mg/kg body weight ip) and, starting 2 wk later, were treated with test compounds for 6 wk and then killed, all rats being subjected to a two-thirds partial hepatectomy at wk 3. Carcinogenic potential was scored by comparing the numbers (no./cm2) and areas (mm2/cm2) of induced glutathione S-transferase placental form (GST-P) positive foci in the livers of groups of about 15 rats with those of corresponding control groups given DEN alone. Positive was scored for a significant increase (P < 0.05) in quantitative values of GST-P positive foci, negative for no change or a decrease. Results for the 94 compounds were also compared with previously published data from Salmonella/microsome (Ames) tests and long-term carcinogenicity studies in rats and mice. Of the known liver carcinogens, 14 out of 14 (100%) mutagenic (Ames test) compounds and 10 out of 12 (83%) non-mutagenic compounds gave positive results in our DEN-PH system (mean 92%). Two hepatocarcinogenic peroxisome proliferators did not enhance the development of GST-P positive foci. Carcinogens other than hepatocarcinogens gave fewer positive results (five out of 17, 29%). One of the 13 compounds reported as non-carcinogenic, malathion, gave positive results in the DEN-PH assay, suggesting that this compound is a weak hepatocarcinogen or tumour promoter for hepatocarcinogenesis based on the two-stage hypothesis for carcinogenesis. The present study also provided information regarding the inhibitory potential of nine compounds. The practical usefulness and benefits of the DEN-PH protocol for the rapid screening of carcinogenic agents are discussed.     

Chronic toxicity of sodium nitrite in the male F344 rat

A long-term feeding study was carried out in rats with sodium nitrite. The test substance was administered as part of a reduced-protein diet to groups of 50, 6-wk-old, male F344 rats at dose levels of 0.2 or 0.5% (w/w) sodium nitrite for up to 115 wk. A control group of 20 males received the reduced-protein diet alone. Throughout the study, there was a dose-related decrease in the rates of body-weight gains and a corresponding decrease in body weights among animals fed sodium nitrite in the diet. Food intakes of rats in the low-dose group were slightly raised over most of the study. In the high-dose group, food intakes were reduced during the first month, but thereafter were similar to those of the control group. This reduction in food intake together with the lower body weights in the nitrite-treated animals, may indicate a reduction in food utilization. In the first week of treatment the following haematological parameters were reduced: red blood cell count, haematocrit and haemoglobin concentration. The red blood cell count continued to fall for 8 wk, then slowly returned to normal by wk 52. A dose-related reduction was noted in both the incidence and time of onset of lymphomas, leukaemias and testicular interstitial cell tumours. Leukaemias were only found in animals with lymphoma, indicating an association between the two lesions. Under the conditions described in this study, sodium nitrite was found not to be carcinogenic when fed to rats in the diet for up to 115 wk, but rather that the incidence of tumours was reduced in a dose-related manner, which correlated with a similar trend in body weights.     

Lack of a modifying effect by the diuretic drug furosemide on the development of neoplastic lesions in rat two-stage urinary bladder carcinogenesis

The effect of the diuretic drug furosemide on two-stage urinary bladder carcinogenesis in F344 rats initiated by N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) was investigated with regard to possible promoting activity. BBN was administered at 2 doses, 0.01 or 0.05%, in drinking water for 4 wk, and thereafter furosemide was given by gavage 3 times weekly for 32 wk, 250 mg/kg body weight. Furosemide ingestion induced diuresis with an alkaline, hypotonic urine. No significant difference with regard to incidences of bladder lesions were apparent between furosemide and control groups. The present investigation indicated that neither furosemide nor its related polyuria acted as a promoter in two-stage urinary bladder carcinogenesis.     

Antiobesity effects of Isaria sinclairii by repeated oral treatment in obese Zucker rats over a 4-month period

In the present study, the antiobesity effects of Isaria sinclairii (I. sinclairii, a fungus cultured on silkworms) powder were investigated in obese (fa/fa) Zucker rats over 4 mo. Rats were given 5 or 10% (w/w) I. sinclairii powder (I. S.), 10% mulberry leaf powder, or 10% silkworm powder mixed with standard diet; a fifth (control) group was given standard diet alone. Dose-dependent decreases in rate of body weight gain were observed in the IS-treated groups after 2 wk of treatment. Interestingly, weights of abdominal adipose tissues surrounding the epididymides were markedly reduced by I. S., in parallel with an attenuated body weight gain. However, no significant differences were observed versus the control group in terms of urinalysis or ocular or histopathological examinations. In the serum, total cholesterol, triglyceride, bilirubin, and low-density lipoprotein (LDL) were significantly lower in the 10% I. S. group than control after 17 wk of treatment. The mulberry leaf diet (10%) significantly reduced serum bilirubin levels. Obese (fa/fa) Zucker rats displayed markedly elevated serum leptin levels (>24.6%) in the I. S. 5 and 10% groups compared with nontreated controls. Data suggest that I. sinclairii exerts an antiobesity effect in Zucker obese rats.     

Suppressing effect of croton oil on intestinal carcinogenesis induced by methylazoxymethanol acetate in rats

The effect of croton oil on intestinal carcinogenesis by methylazoxymethanol acetate (MAM) was examined in ACI/N rats. Twenty seven male and 28 female ACI/N rats were given a single intragastric intubation of MAM at a dose of 25 mg/kg body weight, followed by croton oil at 0.25 ml/kg body weight, 3 times a week, by gastric intubation until the termination of this experiment (365 days). The animals had diarrhea with administration of the croton oil, but the diarrhea had no effect on their gain in weight. Rats from all groups surviving more than 216 days were counted as effective animals. Seventeen out of 54 effective rats which were treated with MAM and croton oil developed intestinal tumors and the incidence of the intestinal tumors was significantly less than that of the group treated with MAM alone (30 out of 50 rats, P less than 0.01). The average number of tumors per rat in the experimental group which was treated with MAM and croton oil (0.6 +/- 1.1) was also smaller than that in the group which was treated with MAM alone (1.0 +/- 1.8), although the difference was not significant. These results suggest that croton oil may suppress some tumor growth at the proper dose in intestinal carcinogenesis which is initiated by MAM.     

Pharmacological studies on experimental nephritic rats (13). The development and aggravation of nephritis due to the repeated administration of liquoid (author's transl)

In order to elucidate the role of blood coagulation system in the development and aggravation of glomerulonephritis, liquoid (Liq) was repeatedly administered to normal or nephritic rats. When Liq 10 mg/kg was given i.v. daily X 22 to normal rats (group I), the urinary excretions of protein and N-acetyl-beta-glucosaminidase and urea nitrogen content did not significantly change as compared with those in the normal control group. In rats given Liq 10 mg/kg i.v. either every 3 days X 8 (group III) or every day X 22 (group IV) from the 15th day after the i.v. administration of anti-rat glomerular basement membrane rabbit serum (AGS) [0.5 ml/150g body weight], these biochemical parameters were not significantly different from those of nephritic control rats given AGS only (group II). The deposits of fibrin or fibrinoids in glomeruli of groups I, II, III and IV, examined by a fluorescence antibody technique were evident in 2, 2, 8 and 10, respectively, out of 10 rats in each group, although the degree of deposition was slight. Under light microscopy, the adhesion between the glomerular capillary wall and Bowman's capsule, hypercellularity, crescent formation and hyalinization were demonstrated in a part of glomeruli, even in group I. concerning the influence of Liq in nephritic rats, the most prominent glomerular change was hyalinization. While in group II the hyalinization was evident in only 17% of glomeruli, in groups III and IV the hyalinization was 41 and 55%, respectively. Although no significant difference was seen between groups II and III regarding other glomerular changes except for hypercellularity, these changes in group IV increased as compared with those in group II. However, hypercellularity was less in groups III and IV than in group II. A slight occlusion of the glomerular capillary lumen was observed, even in group I. In nephritic groups, the degree of the capillary lumen occlusion in group IV was greater than that in group II. From these results, the acceleration of intraglomerular blood coagulation is considered to be a major factor in the development and aggravation of glomerulonephritis.     

Threshold dose of liver tumor promoting effect of β-naphthoflavone in rats

To determine the threshold dose of β-Naphthoflavone (BNF) that induces hepatocellular tumor promoting effects, reactive oxygen species (ROS) generation and thiobarbituric acid-reactive substance (TBARS) formation, and drug-metabolizing enzymes that protect against ROS generation, two-stage liver carcinogenesis model was used. Partial hepatectomized rats (n = 11 to 12) were fed diets containing 0, 0.03, 0.06, 0.125 or 0.25% BNF for 6 weeks after an intraperitoneal injection of N-diethylnitrosamine (DEN) to initiate hepatocarcinogenesis. Histopathologically, glutathione S-transferase placental form (GST-P)-positive foci significantly increased in rats given 0.25% BNF. No marked changes in ROS production and TBARS contents were observed between the BNF treated and DEN alone groups. Real-time RT-PCR showed that the expression of Cyp1a1, Cyp1a2, Cyp1b1 and Nqo1 significantly increased in the groups given 0.03% BNF or more, but Ugt1a6, Akr7a3 and Gstm1 significantly increased in the groups given 0.125% BNF or more. Gpx2 and Yc2 significantly increased in the groups given 0.06% BNF or more and 0.25% BNF, respectively. Inflammation-related genes such as Ccl2, Mmp12, Serpine1 and Cox-2 significantly increased in the 0.25% BNF group. In immunohistochemistry, the number of cyclooxygenase-2 (COX-2)-positive cells increased in rats given 0.25% BNF. These results suggest that 0.25% BNF is the threshold dose for liver tumor promotion, and the fact that inflammation-related genes and COX-2 protein increased in the 0.25% BNF group strongly suggests that inflammation is involved in the liver tumor promoting effect of BNF in rats.     

Multigeneration reproduction study of isomalt in rats

The sugar replacer isomalt was fed to Wistar rats of both sexes throughout three successive generations at concentrations of 0, 2.5, 5 and 10% in the diet. A group of rats fed a diet containing 10% sucrose served as an additional control group. The initial mating was of 100 rats of each sex in each group. For subsequent matings 20 rats of each sex from each group were used. For each generation two litters were reared until they were at least 3 wk old. Feeding isomalt to rats for three successive generations did not induce any adverse effects on fertility, reproductive performance or development compared with control animals fed diets containing maize starch and sucrose instead of isomalt. The second litter of third-generation rats was given detailed gross and microscopic examinations 4 wk after weaning. A marked treatment-related change was an increase in the relative weight of the caecum (filled and empty), 4 wk after weaning in the second litter of third-generation rats fed 10% isomalt. There was also an increase in the relative weight of the filled caecum in males of the 5% isomalt group. These findings were not accompanied by diarrhoea or histological changes in the caecum. The results of the present study did not demonstrate any deleterious effects on the reproduction, maternal performance or development of rats fed isomalt at dietary levels of up to 10% over three successive generations.     

Carcinogenicity study in rats of phytic acid 'Daiichi', a natural food additive.

The carcinogenicity of phytic acid 'Daiichi' (PA), a natural food additive, was examined in Fischer 344 rats of both sexes. PA was added to the drinking-water of groups of 60 male and 60 female rats at levels of 1.25 or 2.5% for 100-108 wk. There was a dose-dependent reduction in the mean final body weights of rats treated with PA. Necrosis and calcification of the renal papillae were observed in PA-treated rats, but not in the controls. The incidences of necrosis (calcification) were as follows: one (three) out of 57 males given 2.5% PA; one (none) out of 59 males given 1.25% PA; 10 (17) out of 55 females given 2.5% PA; six (six) out of 58 females given 1.25% PA. Renal papillomas occurred in three of the high-dose male rats, four of the high-dose female rats, and three of the low-dose female rats. The development of papillomas seemed to be related to calcification and necrosis of the renal papillae induced by PA. While many other tumours developed in all groups, including the controls, the organ distribution of these neoplasms and their histological characteristics did not differ significantly from those known to occur spontaneously in this strain of rats.     

Chronic toxicity/carcinogenicity studies of cocoa powder in rats

Cocoa powder (CP) was fed at levels of 0.0 (control), 1.5, 3.5 and 5.0% for 104 wk to male and female Sprague-Dawley rats derived from the F3b generation of a multigeneration study using the same CP diets. Initial methylxanthine intake was high in all treatment groups, but steadily declined until wk 26. The high dose level provided a mean methylxanthine intake of approximately 57 mg/kg body weight/day for males and 74 mg/kg body weight/day for females from wk 26 to wk 104 of the study. Compared with controls, the historical trend of methylxanthine-associated growth stimulation was evident in rats consuming diets containing 1.5% CP, while body weight was reduced in rats consuming diets containing 3.5 and 5.0% CP. Survival rates were similar in control and CP-fed rats. No evidence of treatment-related clinical disease or ocular effects was noted. An increased incidence of bilateral testicular atrophy and aspermatogenesis was present in males consuming diets containing 5.0% CP. Non-suppurative myocarditis and interstitial fibrosis of the heart were also increased in incidence in both sexes receiving diets containing 5.0% CP. The overall incidences of both pelvic dilatation and renal pelvic microcalculi were increased in most treatment groups. Although there was no difference in the incidence of benign mammary gland fibroadenomas in female rats between the control group and any CP-fed group, a marginally significant (P = 0.04) trend test was apparent. The significance of this finding is doubtful, since the incidence of this lesion in the highest dose group was well within the historical control range for this strain of rats. No evidence of carcinogenicity from dietary CP was found in either sex.     

Liver tumor promoting effect of etofenprox in rats and its possible mechanism of action

To investigate the liver tumor-promoting effects of etofenprox (ETF), a pyrethroid-like insecticide, 6 week-old male F344 rats were given an intraperitoneal injection of N-diethylnitrosamine (DEN). After 2 weeks from the DEN treatment, 12 rats per group received a powdered diet containing 0, 0.25, 0.50, or 1.0% ETF for 8 weeks. At the time of 2nd week of ETF administration, all animals were subjected to two-thirds partial hepatectomy (PH). One rat per group except for the 0.25% ETF group died due to surgical operation of PH. The number and area of glutathione S-transferase placental form (GST-P) positive foci significantly increased in the livers of DEN-initiated rats given 0.50% and 1.0% ETF compared with the DEN-alone group. Quantitative real-time RT-PCR analysis revealed that the mRNA expression of phase I enzymes Cyp2b1/2, phase II enzymes such as Akr7a3, Gsta5, Ugt1a6, Nqo1 significantly increased in the DEN+ETF groups. The immunohistochemistry showed the translocation of CAR from the cytoplasm to the nuclei of hepatocytes in the ETF-treated groups. Reactive oxygen species (ROS) production increased in microsomes isolated from the livers of ETF-treated rats, and thiobarbituric acid-reactive substances (TBARS) levels and 8- hydroxy-2-deoxyguanosine (8-OHdG) content significantly increased in all of the ETF-treated groups and DEN+1.0% ETF group, respectively. The results of the present study indicate that ETF has a liver tumor-promoting activity in rats, and suggest that ETF activates the constitutive active/androstane receptor (CAR) and enhances microsomal ROS production, resulting in the upregulation of Nrf2 gene batteries; such an oxidative stress subsequently induces liver tumor-promoting effects by increased cellular proliferation.     

Promotion of thyroid carcinogenesis by para-aminobenzoic acid in rats initiated with N-bis(2-hydroxypropyl)nitrosamine.

Sulfonamide analogues of para-aminobenzoic acid (PABA), a precursor of folate synthesis, have beneficial effects as antifolate, but thyroid peroxidase inhibition has been reported as a side effect that results in promotion of rat thyroid carcinogenesis. In the present study, effects of PABA itself on F344 rat thyroid carcinogenesis after initiation with N-bis(2-hydroxypropyl)nitrosamine (DHPN) were evaluated. In experiment 1, rats in groups 1-4 received a single subcutaneous injection of DHPN at 2800 mg/kg, and groups 5 and 6 received vehicle saline alone. From 1 week after DHPN initiation, rats in groups 2, 3, 4, and 6 were fed basal diet containing 0.25%, 0.5%, 1.0%, and 1.0% PABA, respectively, for 40 weeks. Rats in groups 1 and 5 received basal diet alone throughout the experiment. The final incidence of thyroid follicular cell adenomas and adenocarcinomas was significantly (p < 0.05 or 0.01) increased in groups 3 and 4 as compared to group 1. No thyroid tumors were found in groups 5 and 6. In experiment 2, animals in group 1 were fed basal diet alone, while groups 2 and 3 were given 0.5% and 1.0% PABA in the diet, respectively, for 2 weeks. Thyroid weights in group 3, and serum thyroid stimulating hormone level and proliferative activity of follicular cells in groups 2 and 3 were significantly (p < 0.05 or 0.01) elevated. In addition, the serum thyroxine level in group 3 was significantly (p < 0.05) depressed. These results clearly indicate that PABA exerts promotion/progression effects on rat thyroid carcinogenesis as a result of hypothyroidism followed by negative-feedback via the thyroid-pituitary axis.     

Effects of lactobacillus,antacids and antibiotics on the levels of nitrite in the gastro-intestinal tracts of rats fed sodium nitrate

No nitrite was detected in the tissues or contents of the gastro-intestinal tracts of normal rats but after 2 wk on a diet containing 0.5% sodium nitrate the levels of nitrite in the levels of nitrite in the stomach, small intestine and large intestine contents were 0.83, 1.64–2.07 and 0.53–0.83 μg/g of contents respectively. Concurrent administration of 2% Lactobacillus preparation and 0.5% sodium nitrate in the diet for 2 wk further increased the nitrite levels in the intestines and slightly increased the level in the stomach, The elevation of nitrite levels induced by sodium nitrate administration was potentiated considerably by combined treatment with sodium bicarbonate and hetacillin producing nitrite levels of 3.16, 2.93–5.18 and 1.96–2.34 μg/g of the contents of the stomach, small intestine and large intestine respectively. Like hetacillin, minomycin and thiamphenicol also potentiated the nitrite production whereas amikacin (another antibiotic) strongly inhibited the formation of nitrite in the stomach. The different effects of the antibiotics may be due to their selective activities on the various microbes. The results indicate that the levels of nitrite in the gastro-intestinal tract are regulated by the level of nitrate intake, the population of microflora and the gastric pH. The safety of combined medication with antacids, antibiotics and Lactobacillus preparations in man deserves further investigation.     

Effects of dietary soya bean trypsin inhibitor concentrate on initiation and growth of putative preneoplastic lesions in the pancreas of the rat

Two studies evaluated the effects of soya bean trypsin inhibitor concentrate (STIC) on early stages of pancreatic carcinogenesis in Wistar rats. In experiment 1, the effects of a 3-month administration of diets containing 3.7% STIC were compared with the effects of administration of diets containing 20% corn oil, in rats pretreated with a single azaserine injection sufficient to initiate putative preneoplastic atypical acinar cell foci. Experiment 2 investigated the capacity of STIC to initiate pancreatic carcinogenesis. Diets containing 3.7% STIC were fed for 4 wk, then diets containing either 5 or 20% corn oil were fed for 3 months. Pancreases were quantitatively evaluated for foci. All groups of azaserine-initiated rats had large numbers of atypical acinar cell foci per cm3 of pancreas. Of these, the group fed 3.7% STIC had pancreatic foci that occupied a significantly greater (P less than 0.01) percentage volume of pancreas than did groups fed 20% corn oil or control diets, which contained 5% corn oil and no added trypsin inhibitor. Very few or no foci were observed in all other groups of either experiment 1 or 2. STIC had a much greater effect on the growth of azaserine-induced lesions than did corn oil. STIC alone did not appear to initiate pancreatic lesions.     

Nelumbo nucifera leaf extract treatment attenuated preneoplastic lesions and oxidative stress in the livers of diethylnitrosamine‐treated rats

Lotus (Nelumbo nucifera Gaertn) possesses antioxidant, hepatoprotective, and anticancer potential. This study determined the protective role of aqueous extract from Nelumbo nucifera leaves (NLE) against N‐diethylnitrosamine (DEN)‐induced oxidative stress and hepatocellular carcinogenesis in a sample of Sprague–Dawley rats. NLE was fed orally to rats in which hepatic carcinoma was induced with DEN for 12 weeks. Five groups of 12 rats each were used for the study: Group I (control group) rats received distilled water; Group II rats were induced with DEN; Group III rats were induced with DEN and cotreated with 0.5% NLE; Group IV rats were induced with DEN and cotreated with 1.0% NLE; and Group V rats were induced with DEN and cotreated with 2.0% NLE. Clinical chemistry, organ weight, inflammatory marker, protein expression, enzyme, and antioxidant analyses were conducted. NLE administration to rats resulted in significantly decreased levels of serum alanine aminotransferase, aspartate aminotransferase, and albumin, which is indicative of hepatocellular damage, compared with the control group. DEN‐induced oxidative stress was inhibited by NLE and this inhibition was paralleled by decreased lipid peroxides and increased glutathione transferase, superoxide dismutase, catalase, and glutathione peroxidase activity in liver tissues. The status of nonenzymatic antioxidants, such as reduced glutathione, was also found to be increased in NLE‐administered rats. Furthermore, NLE decreased tumor size, hepatic Rac1, PKCα, and GSTπ expressions compared with the DEN‐only group. Thus, supplementation of NLE reduced the adverse changes that occur because of liver cancer. These results prove that NLE protects against liver carcinogenesis induced because of treatment with DEN through blocking lipid peroxidation, hepatic cell damage, and enhancing the antioxidant defense system.     

Lack of renal tumour-initiating activity of a single dose of potassium bromate, a genotoxic renal carcinogen in male F344/NCr rats.

The renal tumour-initiating activity of potassium bromate (KBrO3), a known genotoxic rat renal carcinogen, was investigated in male F344/NCr rats. 6-wk-old rats were given KBrO3 intragastrically as a single dose of 300 mg/kg body weight, which was confirmed by our preliminary toxicity study as a maximum tolerated single dose for this strain of rat. Starting 2 wk after KBrO3 treatment, groups of 39 rats received either a basal diet or a diet containing 4000 ppm barbital sodium (BBNa) as a promoting regimen and were killed at 30, 52, or 104 wk. Control rats received either dietary BBNa (4000 ppm) or the basal diet alone from wk 2 to 52 or 104 wk. Nephropathy was observed in all rats treated with KBrO3 followed by BBNa at 30 wk and in rats receiving BBNa alone, but not in rats exposed to KBrO3 alone. Dysplastic renal tubular cell foci (DTF), putative preneoplastic renal tubular cell lesions were found associated with nephropathy in rats exposed to KBrO3 followed by BBNa from 47 wk. The incidences and multiplicities of DTF and renal tubular cell tumours observed from 31 to 104 wk revealed no initiating effect of KBrO3 treatment. These results indicate that the KBrO3 dose of 300 mg/kg did not initiate renal carcinogenesis.     

HPLC analysis of the nitrosation products of chlordiazepoxide

Products originated from Chlordiazepoxide (I) hydrochloride/sodium nitrite interaction were analyzed by HPLC. The studied reactions were carried out in diluted hydrochloric acid solutions at pH values ranging between 0.5-5.0. Depending on the reaction pH values and molar ratios it was possible to find and assess variable amounts of the N-nitrosochlordiazepoxide (II), the dihydroquinazoline (III) and the lactam (IV). The highest degree of N-nitrosation was found at pH 3.5. At this pH value the yields of (II) were respectively 54.8% and 18.3% when the drug (I)/nitrite molar ratios were correspondingly 0.41 and 0.25. When the reaction was performed in concentrations which is possible to find in the gastric juice of patients taking (I) together with nitrite-rich foods the yield of (II) at pH 3.5 was 2.5%. Since in the meantime the genotoxicity of (II) was proved, "in vivo" formation of N-nitrosochlordiazepoxide (II) represents a potential risk not to be underestimated.