Induction of Enzyme Activity in Cell Culture: A Rapid Screen for Detection of Planar Poly chlorinated Organic Compounds

Abstract

Induction of aryl hydrocarbon hydroxylase (AHH) activity in rat hepatoma cell line serves as a simple and rapid method to detect minute (pg) amounts of certain classes of compounds, e.g., dibenzo-p-dioxins, dibenzofurans, and biphenyls. This method may provide a quick screen for such substances in extracts from foods prior to chemical identification. AHH activity is measured by conversion of benzo[a]pyrene (BP) to 3-hydroxy BP in homogenized cell extracts from control and treated cultures and is reported as pmol product formed/mg protein/min. Substances screened by this method include polyhalogenated analogs of dibenzo-p-dioxin (24 compounds), dibenzofuran (11 compounds), biphenyl (7 compounds), and extracts from several food sources. Response of the most reactive compound, 2,3,7,8-tetrachlorodibenzop- dioxin (TCDD) was used to prepare a standard curve, and the AHH activity induced by mole doses of test substance is reported as an ED50 response (the estimated dose needed to produce 50% maximum enzyme induction). The AHH activity induced by food extracts is equated to the standard curve and reported as TCDD equivalents. A potent ED50 response in cell culture appears to correlate well with known toxic responses in other mammalian and avian systems for certain test substances. This correlalation suggests that the cell culture enzyme induction method is a useful model for screening food extracts that are suspected to be contaminated with polychlorinated planar substances. A collaborative study would demonstrate the reproducibility of the method.

The relative sensitivity of chicken embryos to yolk‐ or air‐cell‐injected 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin

“…The LD50s and LD100s of other HAHs, reported by other researchers, tend to agree with our results. Unpublished data by Verret (reported by Bradlaw and Casterline [25] and Kubiak et al [26]) indicated that the chick embryo LD50 was approximately 140 pg TCDD/g egg. Allred and Strange [27] estimated the LD50 of air-cell-injected chickens (injected at the start of incubation and sacrificed at E18) to be approximately 240 pg TCDD/g egg.…”

Rapid Assessment of Induced Cytochrome P4501a Protein and Catalytic Activity in Fish Hepatoma Cells Grown in Multiwell Plates: Response to Tcdd, Tcdf, and Two Planar PCBS

“…Studies of the I-SAR for induction of CYP1A-dependent catalytic activities such as aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin O-deethylase (EROD) in rat hepatoma cells have contributed to an understanding of the varied biological potencies of individual PHAH congeners in mammals [8,9]. Such information has supported the use of cell culture bioassays for detecting PHAH in complex mixtures of chemicals extracted from tissues or the environment [10,11].…”

Aryl hydrocarbon receptor‐based bioassays for dioxin detection: Thinking outside the box

“…Therefore, some POP bioassays measure the enzymatic activity of CYP enzymes or the expression of CYP genes at the transcript or protein levels ( Figure 1B). Being economical, the 7-ethoxyresorufin-O-deethylase (EROD) assay was one of the first widely accepted methodology for detecting POPs (Bradlaw & Casterline, 1979 (Schiwy, Braunig, Alert, Hollert, & Keiter, 2015). Relying solely on CYP's activation, the EROD assay is not always reproducible and measurements strongly depend on the cell line or organism used, the non-specific activation of CYP by impurities (leading to false positives) and the substrate inhibition of CYP enzymes or the clearance of EROD/CYP1A1 activators by other enzymes (leading to false negatives) (Sanderson et al, 1996).…”