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

Abstract

17Dioxins are highly toxic environmental persistent organic pollutants. In several 18 countries, their presence was also reported in cow and human milk samples in the 19 range of 0.023-26.46 and 0.88-19.0 pg/gm of fat, respectively. The detection of 20 dioxns in food samples has been relied on several expensive technologies, which do 21 not represent their toxic effects on consumers. However, mammalian cell based 22 bioassays have such potential to detect the toxins, while representing their toxic 23 effects. Therefore, we tried a three-dimensionally (3D) cultured buffalo granulosa cell 24 based RT-LAMP colour reaction for detecting the presence of added model dioxin, 25 2,3,7,8-tetrachlorodibenzodioxin (TCDD), in commercial milk. The 3D spheroids on 26 the fifth day of culture were treated with different concentrations of TCDD (i.e. 0.02-27 20 pg/ml) directly as well as indirectly through milk fat. After 24hrs of treatment, gene 28 expression studies were performed on certain granulosa cell-specific (CYP19A1, 29 ER-beta, FSHR and LHR) and selective TCDD-responsive (CYP1A1, CYP1B1 and 30 AHR) genes to identify the potential dioxin responsive gene for further RT-LAMP 31 reaction. As the AHR expression in 3D cultured buffalo granulosa cells appears to be 32 a potential gene marker for sensing the added TCDD in the milk, a colour based RT-33 LAMP reaction was successfully attempted for its expression. However, future 34 studies are needed to develop a dose-responsive colour reaction by considering the 35 treatment time less than 24 hrs.36