Impact of obesity on the toxicity of a multi-ingredient dietary supplement, OxyELITE Pro™ (New Formula), using the novel NZO/HILtJ obese mouse model: Physiological and mechanistic assessments

Abstract

Herbal dietary supplement (HDS)-induced hepato- and cardiotoxicity is an emerging clinical problem. In this study, we investigated the liver and heart toxicity of HDS OxyELITE-PRO™ New Formula (OEP-NF), a dietary supplement marketed for weight loss and performance enhancement that was recently withdrawn from the market. Using a novel NZO/HlLtJ obese mouse model, we demonstrated that administration of clinically relevant mouse equivalent doses (MED) of OEP-NF produced cardio- and hepatotoxic risks following both short- and long-term administration schedules. Specifically, gavaging female NZO/HlLtJ with up to 2X MED of OEP-NF resulted in 40% mortality within two weeks. Feeding mice with either 1X or 3X MED of OEP-NF for eight weeks, while not exhibiting significant effects on body weights, significantly altered hepatic gene expression, increased the number of apoptotic and mast cells in the heart and affected cardiac function. The degree of toxicity in NZO/HlLtJ mice was higher than that observed previously in non-obese CD-1 and B6C3F1 strains, suggesting that an overweight/obese condition can sensitize mice to OEP-NF. Adverse health effects linked to OEP-NF, together with a number of other hepato- and cardiotoxicity cases associated with HDS ingestion, argue strongly for introduction of quality standards and pre-marketing safety assessments for multi-ingredient HDS.

Keywords: Cardiotoxicity; Hepatotoxicity; Herbal-induced liver injury; New dietary ingredient; OXYElite pro; Phytochemicals.

Fig. 1.

Analysis of OEP-NF toxicity in a two week gavage study. A) Survival of female NZO/HlLtJ mice gavaged with 2X MED (Day 1) and 1X MED (Day 2–12); B) Body weight dynamics; C) Liver to body weight ratio; D) Serum plasma concentrations of alanine (ALT) and asparate (AST) aminotransferases. Data are presented as means ± SEM. Asterisks (*) denote significant (p < 0.05) difference from control (n=11 for Vehicle group, n=15 for OEP group, except for ALT and AST where n=3 for vehicle and n=10 for OEP). OEP – OxyELITE Pro™ – New Formula.

Fig. 2.

Molecular alterations in the livers of NZO/HlLtJ mice gavaged with OEP-NF for two weeks. A-C) mRNA levels of Avpr1a, Lgr5, and Skil; D) Levels of miR-101a as measured by RT qPCR. Data are presented as means ± SEM. Asterisks (*) denote significant (p < 0.05) and (**) denote significant (p < 0.01) differences from control (n=5 for Vehicle group, n=5 for OEP group). OEP – OxyELITE ProTM – New Formula.

Fig. 3.

Immunoblot analysis of cardiac protein levels of inflammatory cell markers, anti-oxidant enzymes, and overall protein 4-hydroxynonenal (4-HNE) adducts in NZO/HlLtJ mice gavaged with OEP-NF for two weeks. Density of protein bands was determined with the ImageJ software and expressed relative to Glyceraldehyde 3-phosphate dehydrogenase (Gapdh). Data are presented as means ± SEM (n=4 for Vehicle group, n=6 for OEP group). Asterisks (*) denote significant (p < 0.05) difference from control. OEP – OxyELITE ProTM – New Formula.

Fig. 4.

Analysis of OEP-NF toxicity in an eight week feeding study of female NZO/HlLtJ mice. A) Body weight dynamics; B) Liver to body weight ratio; C-D) Serum plasma concentrations of alanine (ALT) and asparate (AST) aminotransferases. Data are presented as means ± SEM (n=4 for each experimental group). Asterisks (*) denote significant (p < 0.05) difference from control. OEP – OxyELITE ProTM – New Formula.

Fig. 5.

In vivo cardiac structure and function after two and eight weeks of feeding of OEP-NF assessed via ultrasonography. Data are presented as means ± SEM. Asterisks (*) denote significant (p < 0.05) difference from control. Delta (^Δ) denotes significant (p<0.05) difference from the low-dose group (n=4 for each experimental group). OEP – OxyELITE ProTM – New Formula.

Fig. 6.

Histological analysis of the heart after eight weeks of feeding of OEP-NF. A) Cardiac area occupied by collagens; B) Cardiac mast cell density; C) Density of apoptotic cells in the heart. Data are presented as means ± SEM (N=4 for each experimental group). Asterisks (*) denote significant (p < 0.05) difference from control. OEP – OxyELITE ProTM – New Formula.