Effect of Aidi Injection on Expression of Cytochrome P450 Enzyme in DEN-induced Hepatocellular Carcinoma Rats / 中国实验方剂学杂志

Objective:To study the effect of Aidi injection （AD） on the expression of cytochrome P450 isoenzyme 1A2，2E1，3A2，2C11（CYP1A2，2E1，3A2，2C11）mRNA and protein in rats with N-nitrosodiethylamine （DEN） chemically induced primary hepatocellular carcinoma（HCC）. Method:Three healthy SD male rats were randomly selected as the blank group， and the remaining rats were treated with DEN intermittently induced primary hepatocellular carcinoma rat model. After success of the model， the rats were randomly divided into model group and AD group， with 3 rats in each group. The rats in the blank group and model group were intraperitoneally injected with 10 mL·kg^-1 saline， while those in the AD group were intraperitoneally injected with 10 mL·kg^-1AD once a day， a total of 14 d intervention. Real-time quantitative polymerase chain reaction（Real-time PCR） and Western blot were used to detect the mRNA and protein expressions of CYP1A2， CYP2E1， CYP3A2 and CYP2C11， respectively. Result:Real-time PCR results showed that after 14 days of drug administration， compared with the blank group， the mRNA expressions of CYP1A2， CYP2E1， CYP3A2 and CYP2C11 were all down-regulated in para-cancerous tissue （PCT） and cancerous tissue （CT） in model group， and there were significant differences （<italic>P</italic><0.05，<italic>P<</italic>0.01）. Compared with the model group， the mRNA expressions of the four subtype enzyme were significantly down-regulated in PCT in the AD group（<italic>P</italic><0.05，<italic>P<</italic>0.01）， while the mRNA expressions of the four subtype enzyme were significantly up-regulated in CT （<italic>P</italic><0.05）， and the expression was down-regulated overall. Western blot results showed that compared with the blank group， the protein expressions of CYP1A2， CYP2E1， CYP3A2 and CYP2C11 in CT of the model group were significantly down-regulated （<italic>P</italic><0.01）， and the protein expressions of CYP3A2 and CYP2C11 were significantly down-regulated in PCT （<italic>P</italic><0.01）. Compared with the model group， the protein expressions of CYP1A2， CYP2E1， CYP3A2 and CYP2C11 in CT and PCT were down-regulated in the AD group， but the differences were not statistically significant. Conclusion:AD can down-regulate the mRNA and protein expressions of CYP1A2， CYP2E1， CYP3A2 and CYP2C11 in rat liver tissues. In clinical use of AD， attention should be paid to drug interactions that may be caused by CYP450 enzyme inhibition.

Effect of Postnatal Exposure to N Nitrosodiethylamine on the Myenteric Plexus of Rat.

Background: Humans are continuously exposed to the different types of nitrosamines found in the diet, drinking water, tobacco smoking, and work place. These are the potential source of exposure in the present population. Nitrosamines are found mainly in cured meat products, smoked preserved foods, beer, whiskey, pickled and salty preserved food materials. Nitrosamines have cytotoxic, carcinogenic and mutagenic properties. Nitrosamines exert toxic or mutagenic effects by promoting DNA damage, oxidative stress and reactive oxygen species formation that causes increased lipid peroxidation, adduct formation, and pro-inflammatory cytokine activation. Increased chronic exposure of low doses of nitrosamines is unavoidable in current environmental conditions. The nitrosamine explored in this study is N-Nitrosodiethylamine (NDEA), representing environmentally significant nitrosamine. Materials and Methods: The present study was conducted on pups of wistar rats, (Rattus norvergicus). Six pregnant wistar rats having same pregnancy time were taken. After delivery sixteen pups were chosen randomly. The control and the experimental groups had eight pups each. Sterile water and NDEA were given as 0.2mg/kg intraperitonea daily to the control and the experimental groups of rat pups respectively, from postnatal day 1 to postnatal day 20. All the rat pups were sacrificed on postnatal day 21 to obtain the tissues of the gastrointestinal tract. Results: A significant reduction of morphometric parameters such as the area, the perimeter and the ferret diameter of the perikaryon of the myenteric neurons of the experimental group found .The number of the myenteric neurons per unit area of muscularis externa was also significantly reduced in the NDEA treated wistar rat pups. Conclusions: Chronic low-level exposure of N-Nitrosodiethylamine (NDEA) caused significant effect on the histoarchitecture of myenteric plexus of wistar rats.

Protective effect of Genistein against N-nitrosodiethylamine (NDEA)-induced hepatotoxicity in Swiss albino rats / 药物分析学报

In the present study, we studied the effect of Genistein against the hepatotoxicity induced by N-nitrosodiethylamine (NDEA). NDEA is present in almost all kinds of food stuff and has been reported to be a hepatocarcinogen. The male rats were exposed to NDEA (0.1 mg/mL) dissolved in drinking water separately and along with 25, 50, 100 mg/mL of Genistein for 21 days. The activities of serum glutamic oxaloacetic transaminase (SGOT), serum glutamic pyruvic transaminase (SGPT), alkaline phosphatase (ALP) and lactate dehydrogenase (LDH) were measured in blood serum. Lipid peroxidation, protein carbonyl content, micronucleus frequency and DNA damage (Comet assay) were performed on rat hepatocytes. The results of the study reveal that the treatment of NDEA along with Genistein showed a significant dose-dependent decrease in the levels of blood serum enzymes i.e., SGOT, SGPT, ALP and LDH (Po0.05). The HE staining of histological sections of the liver also revealed a protective effect of Genistein. A significant dose-dependent reduction in the lipid peroxidation and protein carbonyl content was observed in rats exposed to NDEA (0.1 mg/mL) along with Genistein (Po0.05). The results obtained for the comet assay in rat hepatocytes showed a significant dose-dependent decrease in the mean tail length (Po0.05). Thus the present study supports the hepatoprotective role of Genistein.

Inhibitory Effects of Pharbitidis Semen on Rat Hapatoma Induced by N-nitrosodiethylamine / 医药导报

Objective To study the inhibitory effect of Pharbitidis Semen on rat hepatoma induced by N-nitrosodiethylamine ( NDEA) . Methods SD rats were divided into normal control group, model control group and Pharbitidis Semen group. In model control group and Pharbitidis Semen group, 0. 01% NDEA was applied for 90 days to induce hepatoma, and rats in Pharbitidis Semen group concomitantly received feed containing 6% Pharbitidis Semen at the dosage of 40 g·kg-1 ·d-1 . Thirty days after the hepatoma inducement and Pharbitidis Semen administration, the rats were sacrificed to observe the pathological changes in liver, number of hepatoma nodules and liver weight. The changes of liver/body weight, serum alanine aminotransferase (ALT), γ-glutamyl transferase (γ-GT), and alkaline phosphatase (ALP) were compared. One-way ANOVA (LSD Test) was employed for statistical analysis. Results In the normal control group, the number of hepatoma nodules was 0. 0±0. 0, the liver weight was (9. 87±1. 30) g, the ratio of liver/body weight was (2. 62±0. 24)% and the level of serum ALT was (64. 10±12. 71) U·L-1,γ-GT was (0. 80± 0. 42) U·L-1, and ALP was (121. 20±37. 57) U·L-1. In the model control group, the number of hepatoma nodules was (27. 4±9. 5), the liver weight was (21. 38±7. 29) g, the ratio of liver/body weight was (5. 82±2. 31)%, the level of serum ALT was (175. 70±48. 75) U·L-1, γ-GT was (41. 80±15. 38) U·L-1, and ALP was (200. 50±35. 78) U·L-1. In the Pharbitidis Semen group, the number of hepatoma nodules was (8. 6± 5. 3), the liver weight was (13. 91±3. 55) g, the ratio of liver/body weight was (3. 86±0. 76)% and the level of serum ALT was (113.10±45.35) U·L-1, γ-GT was (13. 40± 6. 15) U·L-1, and ALP was (155. 80±30. 26) U·L-1. The results showed that all indices of Pharbitidis Semen group were higher than those of the normal control group, and lower than those of the model control group (P<0. 01 or P<0. 05). Conclusion Pharbitidis Semen can reduce NDEA-induced injury to the liver cells, and inhibit the overgrowth of the hepatoma.

Alteration in Levels of Minerals in DEN induced Hepatocellular carcinoma in Wistar Albino Rats.

The present attempt has been made to evaluate, and examine the levels of minerals in serum and liver in DEN induced hepatocellular carcinoma in wistar albino rats for possible chemopreventive effect. In hepatocellular carcinogenesis complications such as hepatic fibrosis and cirrhosis may lead to several abnormalities in mineral metabolism, hence attempt is made to evaluate on the level of minerals. Hepatic cancer was induced by single dose of intraperitoneal injections of DEN (200mg/kg body weight) followed by phenobarbital of 0.05% mixed with drinking water for 20 weeks. Concentration of calcium, magnesium, sodium and potassium were assessed in the serum and liver at the end of experimental period. Negative correlations were observed between liver function tests and serum mineral levels, except with albumin. Calcium, magnesium, potassium and sodium concentrations in the serum were decreased after the induction of hepatic cancer. The liver calcium content was increased after DEN treatment. No change occurred in liver sodium content. However, magnesium and potassium content was significantly reduced in the hepatic tissue. The results suggest that in DEN-induced hepato cellular carcinoma alteration of essential elements is noted. The low levels of albumin and the related ascites may be one of the major causes of the imbalance of mineral metabolism in hepatocellular carcinoma. Abbreviations: N-Nitrosodiethylamine (DEN), Hepatocellular carcinoma (HCC), Captain Srinivasa Murti Drug Research Institute for Ayurveda (CCRAS), Tamilnadu Veterinary and animal Science University (TANUVAS), Institutional Animal Ethics Committee (IAEC).

Anti-carcinogenic potential of Euphorbia neriifolia leaves and isolated flavonoid against N-Nitrosodiethylamine-induced renal carcinogenesis in mice.

Anti-carcinogenic potential of hydro-ethanolic extract of Euphorbia neriifolia (EN) leaves and an isolated flavonoid (ENF) was investigated against N-Nitrosodiethylamine (DENA)-induced renal carcinogenesis in mice. Experimental mice were pretreated with 150 and 400 mg/kg body wt of EN, 0.5% and 1% mg/kg body wt of butylated hydroxylanisole (BHA) as a standard antioxidant and 50 mg/kg body wt of ENF for 21 days prior to the administration of a single dose of 50 mg/kg body wt of DENA. Levels of renal markers (urea and creatinine), xenobiotic metabolic enzymes (Cyt P450 and Cyt b5), lipid peroxidation (LPO), antioxidants (SOD, CAT, GST and GSH) and other biochemical parameters — AST, ALT, ALP, total protein (TP), and total cholesterol (TC) were measured to determine the renal carcinogenesis caused by DENA. DENA administration significantly (p<0.001) decreased the body weight and increased the tissue weight. It significantly (p<0.001) enhanced the levels of Cyt P450, Cyt b5 and LPO and decreased the levels of SOD, CAT, GST and GSH content. The activities of AST, ALT and ALP and the TP content and renal markers were also significantly decreased (p<0.001), while TC level was markedly increased after DENA administration, as compared with the normal control group (p<0.001). Pretreatment with EN and ENF counteracted DENA-induced oxidative stress (LPO) and exerted its protective effects by restoring the levels of antioxidants (SOD, CAT, GST and GSH), biochemical parameters (AST, ALT, ALP, TP and TC), renal markers (urea and creatinine) and xenobiotic enzymes (Cyt P450 and Cyt b5) in renal tissue. In conclusion, the present study showed significant anti-carcinogenic potential of the hydro-ethanolic extract of E. neriifolia and ENF against DENA-induced renal carcinogenicity.

Dose-response effects of Elephantopus scaber methanolic extract on N-nitrosodiethylamine-induced hepatotoxicity in rats / 中国天然药物

AIM@#A decoction of Elephantopus scaber (Asteraceae) root is used to treat liver disorders in Indian and Chinese traditional medicine. The study was designed to examine the dose response effects of E. scaber methanolic extract on rats exposed to N-nitrosodiethylamine (NDEA) induced hepatotoxicity (0.02% NDEA in water five days per week, per oral) in preventive and curative models.@*METHODS@#In preventive groups, NDEA was administered for six weeks. Daily doses of E. scaber methanolic extract (200 and 100 mg·kg-1) started one week before the onset of NDEA intoxication and continued for six weeks. In curative animals, NDEA was administered for six weeks followed by treatment with the methanolic n-hexane extract of E. scaber (200 and 100 mg·kg-1) for ten days.@*RESULTS@#E. scaber extract treatment significantly (P ≤ 0.05) reduced the levels of AST, ALT, and MDA in both experimental groups. The extract also enhanced the antioxidant enzyme and protein levels in rats intoxicated with NDEA. Treatment with the extract dose dependently protected the liver from NDEA-induced hepatotoxicity with normal hepatocytes and uniform sinusoids, but in some areas showed degenerating hepatic cells in both treatment groups.@*CONCLUSION@#E. scaber methanolic extract dose dependently prevented and reversed the hepatotoxicity induced by NDEA in both experimental models.

Synergistic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin and N-nitrosodiethylamine on cell malignant transformation / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>The present paper aims to investigate the effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and N-nitrosodiethylamine (DEN) on tumorigenesis and its potential mechanism.</p><p><b>METHODS</b>The potentials of TCDD and DEN in separation or in combination to induce malignant transformation were tested in Balb/c 3T3 cells by using a cell transformation assay method. The possible mechanism of observed effects was studied further by adding α-naphthoflavone (α-NF), a competitive binding agent of TCDD, to the Aryl hydrocarbon receptor (AhR) pathway. The mRNA expressions of Cyp1a1 and Cyp2a5 gene in Balb/c 3T3 cells treated by DEN and TCDD in separation or in combination with or without presence of α-NF were measured with fluorescence quantification RT-PCR technique.</p><p><b>RESULTS</b>The cell transformation frequency (TF) was significantly higher in case of induction with TCDD in combination with DEN, as compared to that with either TCDD or DEN alone. These effects were not inhibited via α-NF. The mRNA expression levels of both Cyp1a1 and Cyp2a5 were enhanced by TCDD treatment alone, but this inducible effect was blocked in cells treated by TCDD and DEN in combination.</p><p><b>CONCLUSION</b>TCDD and DEN had a significant synergistic effect on tumorigenesis when they were used in combination. AhR pathway may not be the key mechanism of this synergistic effect. Thus, it is necessary to further test the potential mechanism involved in cancer development.</p>

Cancer promoting effects of N-nitrosodiethylamine (DEN) and 2-acetylaminofluorene (2-AAF) with medium term rat liver bioassay / 中国药理学通报

AIM To study the cancer promoting effects of N nitrosodiethylamine (DEN) and 2 Acetylaminofluorene (2 AAF). METHODS Medium Term Rat Liver Bioassay (MTRLB). Male SD rats were initially given a single dose (200 mg?kg -1 ) of DEN ip and starting 2 weeks later, were treated with 10, 33 and 100 ppm DEN in drinking water, or with 2 2, 6 6 and 22 mg?kg -1 2 AAF by gavage for 6 weeks. All rats were subjected to two thirds partial hepatectomy at week 3 and killed at the end of week 8. Carcinogenic potential was scored by comparing the numbers and areas in induced glutathione S transferase placental form (GST P) positive foci in the liver with those of corresponding control group given DEN alone. RESULTS Both DEN and 2 AAF caused the increases of the numbers and areas of GST P positive foci in the liver, and showed dose response relationship. CONCLUSION Both DEN and 2 AAF shows cancer promoting effects, and MTRLB was a convenient, economical and effective tool to study the cancer promoting effects of test chemicals.