Study On The Synergistic Effect Of Environmental Carcinogens And Its Mechanism

The higher incidence of tumor in Huaihe River basin has become a serious public health event. The water pollution may contribute to this event. So, to evaluate the relativity between water pollution and tumorigenesis is required. A polluted water sample is a mixture that contains various types of potential carcinogens, such as chemicals and virus. These carcinogens often cooperate to cause cancer. Therefore, it is necessary to study this synergistic effects and their mechanism, and to assay the carciongenic potency of waters using an appropriated method.Cell transformation has been defined as the induction of certain phenotypic alterations in cultured cells that are characteristic of tumorigenic cells. It has been shown to be a multistage process which closely models the various stages of in vivo carcinogenesis. Cell transformation assay can provide some crucial evidence specific to the tumorigenic potential of various types of carcinogens, which cannot be supplied by genotoxicity testing. In this paper, an improved cell transformation assay protocol was established by selecting appropriate cell cultures media, concentration of fetal bovine serum (FBS), administration ways and program of cell transformation. Using this improved method, the carcinogenic potencies of diethylnitrosamine (DEN), aflatoxin B₁ (AFB₁) and microcystin-LR (MC-LR) were examined. Synergistic effects between DEN and MC-LR, DEN and 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) were tested as well. The results show that any of DEN, AFB1 and MC-LR can induce cell malignant transformation significantly and show dose-response relationships. When administered together, the tranforamtion frequency induced by DEN plus TCDD are three times higher than that induced by DEN alone, and 15 times higher than TCDD. But for DEN plus MC-LR, the transformation frequency was decreased when they worked together than MC-LR alone.Then, three water samples, one river water sample and two well water samples, from Huaihe River basin were tested. The River water sample, even without being condensed, can induce significantly higher cell transformation frequency than control, and the transformation frequency increase with the increase of concentration multiple. The water sample from shallow well can induce a significantly higher transformation frequency when it is condensed at least 100 times, however, the water sample from a deeper well doesn't show the ability to induce cell malignant transformation.The mechanism of the synergistic effect between DEN and TCDD was explored as we found this effect between them. We speculated that TCDD up regulated the expression of cytochrome P450 oxygenases which can metabolize DEN to its ultimate carcinogen form. According to this speculation, the AhR inhibitorα-NF will inhibit the synergistic effect between TCDD and DEN. However, the cell transformation inhibition assay shows thatα-NF doesn't work. The results of real time RT-PCR show that the transcription level of Cyp 1a1 and Cyp 2a5 gene are up regulated by TCDD alone, but down regulated after DEN was administer together with TCDD.From these results above we can conclude that: (1) The improved Balb/c 3T3 cell transformation assay is reliable and time-saving, and can be efficiently used in the study of synergistic carcinogenic effects among carcinogens, and the carcinogenic test of water sample. (2) MC-LR, DEN and AFB₁ each have carcinogenic potency. TCDD have little ability to induce cell malignant transformation, but it is a potent cocarcinogen with DEN. Different to the result of two-stage model, there exist no synergistic effect between DEN and MC-LR when they are administered at the same time. (3) The river water of Huaihe basin has the ability to induce tumor. The carcinogenicity of Well water is weaker than surface water, and reduces with the increase of well depth. This conclusion needs to be verified by assaing more water samples. (4) The interaction between DEN and TCDD isn't the result that TCDD enhance the expression of cytochrome P450-linked DEN-bioactiving oxygenases. The mechanism may involve in more complex regulation procession which is worthy of a closer study.