Modification by curcumin of mutagenic activation of carcinogenic N-nitrosamines by extrahepatic cytochromes P-450 2B1 and 2E1 in rats

Abstract

To elucidate the mechanism underlying suppression by curcumin of esophageal carcinogenesis induced by NMBA, we evaluated the CYP level and mutagenic activation of environmental carcinogens, by immunoblot analyses and Ames preincubation test, respectively, and bilirubin, 4-nitrophenol and testosterone UDPGT activities in F344 rats treated with curcumin and/or NMBA. No significant alterations in the hepatic levels of constitutive CYP proteins, mutagenic activation by liver S9 or hepatic UDPGT activities were produced by subcutaneous treatment with 0.5 mg/kg NMBA for 5 weeks and/or feeding of 0.05% and 0.2% curcumin for 6 weeks. In contrast, gavage of 0.2% curcumin decreased esophageal CYP2B1 and 2E1 by up to 60%, compared with vehicle control. Similarly, intragastric treatment with 270 mg/kg curcumin decreased esophageal and gastric CYP2B1 and CYP2E1, but not in lung, kidney or intestine. Conversely, large intestinal CYP2B1 was 2.8-fold higher in the treated rats than in control rats. Mutagenic activities of NOC, including NMBA, in the presence of esophagus and stomach S9 were markedly decreased in the treated rats, whereas those in the presence of large intestine S9 were 2.2-3.0-fold above control. These results show that modifying effects of curcumin on esophageal carcinogenesis can be attributed to a decrease in metabolic activation of NMBA by esophageal CYP2B1 during the initiation phase, without the contribution of metabolic activation and inactivation by liver. Further, the present findings suggest the potential of curcumin for modification of gastric and intestinal carcinogenesis initiated with NOC.

Figure 1

Immunoblots and densitometric determination of expression of CYP protein in esophagus (left) and liver (right) microsomes from rats treated with NMBA, curcumin or both. Both microsomes were pooled from five rats treated with vehicle (lane 1) or 0.5 mg/kg NMBA (lane 2) three times per week for 5 weeks, 0.05% (lane 3) or 0.2% (lane 4) curcumin for 6 weeks, and NMBA + 0.05% (lane 5) or + 0.2% (lane 6) curcumin. Lane 7 contains CYP standards from male Sprague–Dawley rats treated with PB (A and D), acetone (B) or MC (C). Liver microsomes contain 0.4 µg (A–C) and 1.0 µg (D) microsomal protein, and the values represent means ± SD of pmol/mg microsomal protein obtained from four experiments. Esophagus microsomes (A, B) contain 30 µg microsomal protein, and the values represent means ± SD of the ratio to arbitrary units obtained with the vehicle group. n.d., not detected.

Figure 2

Immunoblots and densitometric determination of expression of CYP protein in esophagus (left) and liver (right) microsomes from rats treated orally with curcumin or subcutaneously with NMBA as a single dose. Both microsomes were pooled from 10 rats 24 h after each treatment. Lanes PB, acetone and MC contain CYP standards. The amounts of each microsomal protein, including (C) and (D) in the esophagus, and the means ± SD are identical to those described in Figure 1 legend. *P < 0.01, compared with the vehicle group (Student's t‐test). n.d., not detected.

Figure 3

Immunoblots and densitometric determination of CYP2B (A) and 2E (B) expressions in extrahepatic tissue microsomes from rats orally treated with 270 mg/kg curcumin as a single dose. These microsomes were pooled from 16 rats 24 h after treatment with vehicle (V) or curcumin (C). Lanes PB and acetone contain CYP standards and the values represent means ± SD of the ratio to arbitrary units obtained with the vehicle group from between four and eight experiments. The assays of electrophoresis were carried out with 30 µg microsomal protein, except for lung CYP2B1/2 and kidney CYP2E1, for which 5 µg microsomal protein was loaded. *P < 0.05 and **P < 0.01, compared with the vehicle group (Student's t‐test). n.d., not detected.

Figure 4

Mutagenic activities of various carcinogens in the Salmonella typhimurium tester strains TA100 (upper, six NOC) and TA98 (lower, four HCA, BP and AFB₁) in the presence of liver S9 from rats treated with NMBA, curcumin or both. Each test was carried out in duplicate (four to eight plates) with liver S9 pooled from five rats each treated with vehicle (Group 1; clear), NMBA (Group 2; light stippled), 0.05% curcumin (Group 3; hatched), 0.2% curcumin (Group 4; horizontal) or NMBA + 0.2% curcumin (Group 6; solid) for up to 6 weeks. Each bar represents the means ± SD after subtraction of spontaneous revertants (TA100, 108–136; TA98, 9–14). DMN and DEN were assayed in both doses of 1 and *10 mg/plate, and Trp‐P‐2 was in 0.3 µg/plate.

Figure 5

Effect of a single treatment with 270 mg/kg curcumin on the mutagenic activities of NMBA, DEN and DMN in the Salmonella typhimurium strain TA100 in the presence of esophagus, stomach or large intestine S9. Each test was carried out in duplicate (four to six plates) with tissue S9 pooled from 16 rats each treated with vehicle (clear) or 270 mg/kg curcumin (horizontal). Each bar represents the means ± SD after subtraction of spontaneous revertants (111–124). *P < 0.05 and **P < 0.01, compared with the vehicle group (Student's t‐test).