Glyphosate-based herbicides (GHB), which contain glyphosate as the active ingredient, are the most commonly used organophosphorus pesticides worldwide (Benbrook, 2016). With the emergence of weed- and glyphosate-resistant crops, the utilization of GBH has increased sharply in recent decades (Green and Siehl, 2021; Zhu et al., 2022). As a result, glyphosate residues are frequently detected in the air, soil, water, and plants (Maqueda et al., 2017; Soares et al., 2021; Pelosi et al., 2022). Additionally, glyphosate and its major metabolite aminomethylphosphonic acid (AMPA) have also been detected in many food products, such as breakfast cereal, soy protein isolate, coffee, wine, and honey (Ehling and Reddy, 2015; Zoller et al., 2018; de Souza et al., 2021). These events inevitably increase the potential of the exposure in population. Notably, a growing body of literature reports that glyphosate and AMPA have been detected in the blood, urine, and maternal milk of the occupational and general human (Connolly et al., 2018; Parvez et al., 2018; Gillezeau et al., 2019). Therefore, the widespread use of GBH has raised concerns about public health in the last decade.
GBH kills weeds specifically by inhibiting 5-enolpyruvylshikimate-3-phosphate synthase, a central enzyme involved in the biosynthesis of aromatic amino acids in plants and microorganisms but not in humans and animals (Priestman et al., 2005). Therefore, glyphosate is considered harmless to humans, and the European Food Safety Authority has declared that glyphosate is unlikely to be carcinogenic to humans (Greim et al., 2015; European Food Safety, 2017). However, the harmful effects of glyphosate have been debated for decades. Many studies have demonstrated that glyphosate or GBH can increase DNA damage, micronuclei frequencies and chromosomal aberration (Milic et al., 2018; Santovito et al., 2018). Moreover, a recent meta-analysis revealed an association between high cumulative exposure to GBH and an increased risk of non-Hodgkin lymphoma in humans (Zhang et al., 2019). In addition, numerous toxicity studies have reported that GBH has non-target toxic effects on mammalian metabolism, such as neurotoxicity, reproductive toxicity, and hepatotoxicity (Mesnage et al., 2017; Jarrell et al., 2020; Luna et al., 2021).
Glyphosate is poorly metabolized, directly excreted in urine and feces, and rarely accumulates in animals (Soares et al., 2021). In this sense, researchers believe that glyphosate is nontoxic to antidotal organs. However, accumulating evidence suggests that the liver is a major target organ of glyphosate and GBH exposure in rodent models and aquatic animals (Shiogiri et al., 2012; Mesnage et al., 2015). A previous study revealed that GBH exposure causes hepatic pathological alterations and increases the levels of biochemical markers of liver function (Caglar and Kolankaya, 2008). It is believed that the detrimental effect of GBH exposure on the liver is mainly due to the overproduction of reactive oxygen species (ROS) and triggering of oxidative damage by inhibiting enzymatic and non-enzymatic antioxidants in rats (Turkmen et al., 2019). Of note, mitochondria, the major cellular sites of ROS production, have been documented as targets of GBH exposure (Strilbyska et al., 2022). In addition, an increasing number of studies have reported that GBH exposure results in an imbalance of lipid metabolism in the livers of mice and fish (Ford et al., 2017; Liu et al., 2021). Meanwhile, Pandey et al. revealed that short-term exposure to GBH induces glycogen depletion and the progression of fatty liver disease in rats (Pandey et al., 2019). Nevertheless, the molecular regulatory network of energy metabolism disorder due to GBH exposure in the liver has not been well described.
Induction of inflammation is considered an important pathogenic factor in hepatic damage and dysfunction. A previous study reported that GBH exposure could induce inflammatory reactions in the liver of common carp via inflammatory mediators (Ma and Li, 2015). Another study suggested that GBH induced inflammation, represented by an increase in C-reactive protein and cytokines TNF-α, IL-1β, and IL-6 in the rat liver (Pandey et al., 2019). In addition, a recent study confirmed that GBH increased the TNF-α: IL-10 and IFN-γ ratio in serum, leading to an imbalance between proinflammatory and anti-inflammatory cytokines (Ngatuni et al., 2022). It is noted that hepatic inflammation plays a key role in the pathogenesis of nonalcoholic fatty liver disease (Manne et al., 2018). A study has shown that glyphosate excretion is associated with steatohepatitis and advanced liver fibrosis in patients with fatty liver disease (Mills et al., 2020). Therefore, GBH exposure may be associated with the progression of liver inflammation. However, the literature remains sparse and focuses only on cytokines related to the hepatotoxicity of GBH exposure in mammals.
Although previous studies have suggested an association between glyphosate exposure and liver lesions, a systematic investigation has not been conducted to clarify the molecular regulatory network of GBH-induced energy metabolism disorders and inflammatory damage in the liver. In this study, hepatic structure and function indicators, oxidative stress state, transcriptome profiles and key protein alterations were investigated to systematically and mechanistically depict hepatotoxicity in mice after 30 d of RoundUp® exposure. Our study sheds light on the molecular mechanism underlying GBH-induced hepatotoxicity in mice, providing reliable data for the toxicological risk evaluation of glyphosate in mammals.