Effect of Dioxins in Milk on the 3D Cultured Primary Buffalo Hepatocyte Model System

Abstract

Cow and human milk have been reported to contain dioxins ranging from 0.023 to 26.46 and 0.88 to 19 pg/g of fat, respectively. However, the toxic effects of the dioxins in the milk in this range of concentrations were not explored. Therefore, considering the outbred livestock tissues as better models than inbred laboratory animals, the present study targeted to study the effect of dioxins present in the milk on three-dimensionally (3D) cultured buffalo primary hepatocyte spheroids. The spheroids were treated with a model dioxin, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), directly and also through milk fat at different concentrations (i.e, 0.02−20 pg/mL) for 24 h. Among the liver-cell-specific (ALB, HNF4α, and AFP) genes, a similar ALB and upregulated HNF4α expression at all treatments indicated the functional and transcriptionally active hepatocyte spheroids. Supportingly, no significant difference in the antiapoptotic gene expression between the treatments of milk fat and milk fat containing dioxins indicated the survivability of the spheroids during dioxin treatments. Among the selected TCDD responsive (CYP1A1, CYP1A2, AHR, CYP1B1, and TIPARP) genes, a nonsignificant increasing trend of the CYP1A1 expression was observed from 0.2 to 10 pg/mL of TCDD concentration through milk fat. This pattern was similar to the reported insensitive response of human primary hepatocytes toward dioxins than that of rat primary hepatocytes. This may indicate that the buffalo hepatocyte spheroids could be better models than rats for TCDD hepatotoxic studies. Further, TCDD in the milk in the range of 0.02−20 pg/mL concentration may not be very hepatotoxic.

Effect of dioxins in milk on 3D-cultured primary buffalo granulosa cells, a pilot study for a prospective RT-LAMP colour reaction for dioxin toxicity

“…With an assumption that all the extracted fat would recover the added TCDD, total milk fat was extracted from TCDD spiked milk using dichloromethane-ethanol extraction method. The extracted fat was dissolved in 1 ml of 1% DMSO, which was used to treat the granulosa cell spheroids at different concentrations, such as 0.02 pg/ml, 0.2 pg/ml, 2 pg/ml, 10 pg/ml and 20 pg/ml of media (Goud, et al , 2019).…”