Transgenic Zebrafish Expressing Rat Cytochrome P450 2E1 (CYP2E1): Augmentation of Acetaminophen-Induced Toxicity in the Liver and Retina

Abstract

Metabolic activation is the primary cause of chemical toxicity including hepatotoxicity. Cytochrome P450 2E (CYP2E) is involved in this process for many hepatotoxicants, including acetaminophen (APAP), one of the most common analgesics and antipyretics. Although the zebrafish is now used as a model for toxicology and toxicity tests, the CYP2E homologue in zebrafish has not been identified yet. In this study, we prepared transgenic zebrafish embryos/larvae expressing rat CYP2E1 and enhanced green fluorescent protein (EGFP) using a β-actin promoter. Rat CYP2E1 activity was confirmed by the fluorescence of 7-hydroxycoumarin (7-HC), a metabolite of 7-methoxycoumarin that was specific for CYP2 in transgenic larvae with EGFP fluorescence (EGFP [+]) but not in transgenic larvae without EGFP fluorescence (EGFP [−]). APAP (2.5 mM) caused reduction in the size of the retina in EGFP [+] larvae but not in EGFP [−] larvae, while APAP similarly reduced pigmentation in both larvae. APAP at even 1 mM reduced the liver size in EGFP [+] larvae but not in EGFP [−] larvae. APAP-induced reduction of liver size was inhibited by N-acetylcysteine. These results suggest that rat CYP2E1 is involved in some APAP-induced toxicological endpoints in the retina and liver but not in melanogenesis of the developing zebrafish.

The developmental stage is a critical parameter for accurate assessment of the drug-induced liver injury (DILI) potentials of drugs with the zebrafish larval liver model

“…10 mM APAP caused lethality when applied between 2-5 dpf. 2.5 mM APAP caused changes in melanocyte pattern, and 5 mM APAP caused smaller eyes and defects in pigmentation, which are previously reported phenotypes (Figure 1A) (Sato et al, 2023). However, the same concentrations of APAP did not induce any morphological or pigmentation defects when applied between 5-7 dpf (Figure 1B).…”

“…Zhang et al, 2014). Several studies confirmed that drug detoxification enzymes are conserved in zebrafish, and drugs are processed in zebrafish liver (Sato et al, 2023). Early zebrafish between 2-5 dpf are highly preferred for hepatotoxicity studies, this is a stage during which liver specification and maturation take place (Cassar et al, 2019;Chu & Sadler, 2009).…”

Augmentation of Pectoral Fin Teratogenicity by Thalidomide in Human Cytochrome P450 3A-Expressing Zebrafish

“…This pattern of expression of hCYP3As and 1A1 might contribute to diverse malformations in addition to the absence of serious effects on the general development of zebrafish. While more homologous expression of hCYP3A7 in zebrafish may result in serious impacts, it is still feasible to generate transgenic zebrafish expressing this enzyme, as has been accomplished with rat CYP2E1-transgenic fish [ 36 ].…”

"Insights From a Rat Model of Paracetamol-Induced Hepatotoxicity into the Molecular Mechanisms of Silymarin and Resveratrol Combination Therapy for Protecting Liver Function"

“…Additionally, cytochrome P450 inhibitors can thwart PCM activation into its toxic form. Another approach involves using glutathione supplementation to counter PCM-induced hepatotoxicity, as it replenishes depleted glutathione levels [15,16]. Existing treatments for PCM-induced hepatotoxicity have some limitations like NAC has limited efficacy in preventing liver damage caused by PCM overdose [12], regular monitoring of risk factors after getting treatment [17], and limited prevention options after PCM overdose is still not well-established [18].…”