Effects of Glyphosate and Terbuthylazine on Liver Carcinogenesis: Integrating Toxicogenomics and Risk Assessment

• Grant number: 25/05637-4
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)
• Start date: January 01, 2026
• End date: December 31, 2029
• Field of knowledge: Health Sciences - Medicine - Pathological Anatomy and Clinical Pathology
• Principal Investigator: Guilherme Ribeiro Romualdo
• Grantee: Luana Riechelmann Casarin
• Host Institution: Faculdade de Medicina (FMB). Universidade Estadual Paulista (UNESP). Campus de Botucatu. Botucatu , SP, Brazil

Abstract

Glyphosate (GLY) is one of the most widely applied active ingredients, while Terbuthylazine (TBA) has also seen growing usage. Due to their widespread use, residues of these herbicides are frequently detected in soil, air, water, and even food products, potentially leading to human exposure. Despite evidence pointing to the liver as a target organ for the toxicity of these herbicides, studies specifically evaluating their role in hepatic carcinogenesis are still limited. Furthermore, the implications of combined GLY+TBA exposure-potentially leading to "cocktail effects"-are not fully understood and are often overlooked in carcinogenesis research. Thus, this study aims to (A) evaluate the effects of exposure to GLY formulation and/or TBA formulation on hepatic carcinogenesis in adult rats and (B) assess the effects of exposure to the same herbicides on normal hepatocytes (in vitro). To achieve this, five-week-old male rats will receive two weekly doses of the carcinogen Diethylnitrosamine (100 mg/kg body weight, intraperitoneally) or vehicle (0.9% saline solution) for three weeks. In the eighth week, rats will be exposed, five times a week, to (A) a GLY formulation [Roundup Original DI (57.7% w/v GLY diammonium salt) at GLY-equivalent doses of 5, 0.5, or 0.005 mg/kg body weight b.w.]; (B) a TBA formulation [Timber 500 SC (50.0% w/v TBA) at TBA doses of 4, 0.4, or 0.004 mg/kg b.w.]; or (C) a mixture of both herbicides at low (0.005 + 0.004 mg/kg), intermediate (0.5 + 0.4 mg/kg), or high (5 + 4 mg/kg) doses for one week and will be euthanized at week 9 (endpoint #1) (n=6/group). Other animals will continue receiving the herbicides for 16 weeks and will be euthanized at week 32 (endpoint #2) (n=12/group). At endpoint #1, liver samples will be collected for immunohistochemistry (Ki-67 and H2AX), MDA and FRAP quantification, and transcriptome analysis. Blood samples will be collected for comet assay, bone marrow for micronucleus testing, and serum for ALT, AST, and cytokine array analysis. At endpoint #2, liver samples will be analyzed for immunohistochemistry (Ki-67 and GST-P, a preneoplastic lesion marker) and transcriptome profiling. For the in vitro study, normal liver cell lines (THLE-2) in monolayer and spheroid cultures will be exposed to GLY, TBA, or their mixture (at nanogram-range concentrations to be determined) for up to 72 hours. Cell viability, cell cycle progression, genotoxicity, and transcriptomic responses will be evaluated. In both the in vivo (endpoint #1) and in vitro experiments, transcriptomic points of departure (tPoD) will be established and compared with long-term outcomes (endpoint #2), providing insights for future risk assessment using New Approach Methods. The findings of this study may reveal enhanced carcinogenic effects due to herbicide mixtures, prompting a reassessment of current safety standards for herbicide exposure. Additionally, the validation of New Approach Methods could contribute to refining herbicide risk assessment, supporting the revision of regulatory guidelines. (AU)