The Effects Of Long Term Oral Administration Of Aflatoxin G₁ On Mouse Liver Inflammation And Tissues Damage

AIM: Aflatoxin,including AFB₁ and AFG₁,is a strongly carcinogenic mold-produced contaminant of dietary staples in the world.It was well known that Aflatoxin B₁ could induce liver cancer.As a major naturally liver carcinogen,several lines of studies show that AFB₁ is metabolized mainly by cytochrome P450（CYP450）to form AFB₁-DNA adducts,which then cause oxidative DNA damage.Oxidative DNA damage is also an important mechanism involved in the cytotoxicity and carcinogenic effect of AFB₁.It has been shown that pro-inflammatory cytokine TNF-α could upregulate CYP450 involved in liver cell damage,which indicates a relation between inflammatory response and CYP450 activation.Our previous studies showed oral gavage of AFG₁ for 6 months induced chronic lung inflammation,which causes oxidative damage in alveolar epithelial cells evidenced by increasing oxidative damage marker SOD-2 and CYP450 expression.Furthermore,it is showed that TNF-α could upregulate CYP450 expression to enhanced AFG₁-induced DNA damage in A549 cells.The results indicate that upregulation of CYP450 by inflammation may promote the activation of AFG₁,and then enhance oxidative DNA damage.However,whether long term oral administration of Aflatoxin G₁ induces liver inflammation which upregulates CYP450 expression and DNA damage in liver cells is still unknown.In this study,we collected the liver samples from the mice after oral gavage of AFG₁ for 6 months in our previous experiment.We will evaluate whether oral gavage of AFG₁ induces liver inflammation by observing the histology alteration and measuring inflammatory cytokines production.We evaluated the liver cell damage by detecting CYP450-3A4 expression as well as the established markers of oxidative stress-SOD-2 expression.Our results provide a new mechanism of Aflatoxin-induced oxidative damage in liver cell.Method: We collected the liver samples from the mice after oral gavage of AFG₁ for 6 months in our previous experiment.We will evaluate whether chronic AFG₁ exposure through ingestion induces liver inflammation by observing the histology alteration.The expression of inflammatory cytokines,such as MCP-1/CCL-2、CXCL-1、 TNF-α、IL-6、IL-1and TGF-β,in liver tissues will be measured by Real-time PCR.The expression of 3A4 and SOD-2 in liver tissues will be detected by IHC staining.The expression of COX-2,3A4 and SOD-2 in liver tissues was also measured by Western Blot.Results:1 AFG₁ treatment resulted in inflammatory responses and upregulation of cytokines in lung tissuesAfter H&E staining,we observed increased inflammatory cells infiltration in liver tissues of AFG₁-treated mice.The infiltrated cells located close to central vein and protorl area.The real-time PCR results showed that the expression of TNF-α、IL-6、IL-1、MCP-1/CCL-2、CXCL-1 and TGF-β in liver tissues treated with AFG₁ was higher than that in control mice.The results indicate that chronic AFG₁ exposure through ingestion induces liver inflammation.2 The effect of AFG₁ treatment on the expression of COX-2 on liver tisssuesWestern blot result showed that the expression of COX-2 on liver tissues treated with AFG₁ was higher than that in control mice.3 The effect of AFG₁ treatment on the expression of SOD-2 on liver tisssuesIn normal lung tissues,faint immunoreactivity for SOD2 is detected in the cytoplasm of liver cells.However,after AFG₁ treatment,increased SOD2 positive cells were detected in liver tissue of AFG₁-treated mice.Western blot result showed that the expression of SOD-2 on liver tissues treated with AFG₁ was higher than that in control mice.The result indicates that chronic AFG₁ exposure through ingestion induces oxidative damage in liver cells.4 The effect of AFG₁ treatment on the expression of CYP3A4 on liver tisssuesIn normal lung tissues,faint immunoreactivity for 3A4 is detected in the cytoplasm of liver cells.However,after AFG₁ treatment,increased 3A4 positive cells were detected in liver tissue of AFG₁-treated mice.The expression of 3A4 on liver tissues treated with AFG₁ was higher than that in control mice.The result indicates that Aflatoxin G1-induces liver inflammation may activate 3A4,which enhances AFG₁-induced oxidative damage in liver cells.Conclusion:1 Chronic exposure of AFG₁ induces liver inflammation evidenced by increased inflammatory cells infiltration,cytokines production,and COX-2 expression.2 Chronic exposure of AFG₁ increased the oxidative stress marker SOD-2 expression,suggesting Aflatoxin G1-induces liver inflammation may cause oxidative damage in liver cell.3 Chronic exposure of AFG₁ through ingestion promoted 3A4 expression which may enhance AFG₁-induced oxidative damage in liver cells.