Wild C. sinensis (Cordyceps sinensis) is a medicinal fungus belonging to the Clavicpitaceae family that parasitizes the larvae of Lepidopteran insects (Committee, 2020). It is distributed primarily in the high-altitude grassland regions of the Tibetan Plateau (Yi et al., 2011). Traditionally, wild C. sinensis has been recorded for tonifying the kidneys and lungs and for preventing bleeding and resolving phlegm (Committee, 2020). Studies indicate that it possesses immune-modulatory, anticancer, lung and liver-kidney protective, antioxidant, and antiaging effects (Chen et al., 2013; Fu et al., 2019; Ong and Aziz, 2017; Zhang et al., 2021). However, the detection of relatively high arsenic levels (2.10–9.97 mg/kg) in wild C. sinensis (Wang et al., 2008) has raised substantial safety concerns regarding its chronic consumption.

The National Medical Products Administration of China reported that the total arsenic content in wild C. sinensis ranged from 4.4 to 9.9 mg/kg, and long-term consumption may lead to arsenic accumulation in the body, thus increasing health risks (NMPA, 2016). Xiao et al. further detected arsenic levels of 5.77–13.20 mg/kg in wild C. sinensis, reinforcing concerns about arsenic accumulation with chronic intake (Xiao et al., 2021). Chronic arsenic exposure at low doses typically results in progressive bioaccumulation in various organs, particularly causing severe damage to the liver and kidneys. Mechanistically, arsenic accumulation induces oxidative stress, cellular apoptosis, and steatosis in hepatic cells, ultimately resulting in liver dysfunction, cirrhosis, or hepatocellular carcinoma. Renal arsenic accumulation often leads to renal tubular injury, impaired renal filtration, and even chronic renal failure and fibrosis (Abdul et al., 2015; Centeno et al., 2006; Chen et al., 2024; Santra, 2015; Zheng et al., 2014). The toxicity of arsenic is closely related to its chemical form. Organic arsenicals exhibit low toxicity and negligible bioaccumulation. Among inorganic arsenic species, trivalent arsenic (AsIII) has the highest toxicity; however, although it is less toxic, pentavalent arsenic (AsⅤ) still poses significant health risks (Hughes, 2002). Studies have shown that the arsenic in wild C. sinensis is primarily inorganic arsenic, which is more toxic, accounting for 8.69% of the total arsenic, underscoring potential toxicity concerns (Zhou et al., 2018; Zuo et al., 2018).

Given these risks, regulatory authorities worldwide have implemented strict arsenic limits in pharmaceuticals and foods. The Chinese Pharmacopoeia (Committee, 2020) allows a maximum total arsenic content of 2.0 mg/kg in herbal medicines, whereas the European Pharmacopoeia (HealthCare, 2017) sets the limit at 1.5 mg/kg for pharmaceutical products. The International Council for Harmonization of Technical Requirements for Medicinal Products for Human Use (ICH) has established a limit of 0.015 mg/day for oral exposure to inorganic arsenic (ICH, 2022). In addition, the World Health Organization (WHO) recommends a maximum arsenic concentration of 0.01 mg/L in drinking water (WHO, 2011), whereas the European Union and China’s National Food Safety Standards set the limit for inorganic arsenic in rice at 0.20 mg/kg (Commission, 2006; Wu, 2012). The arsenic levels of wild C. sinensis exceed these regulatory thresholds, raising concerns about the potential health risks of long-term consumption. Despite these concerns, most studies have focused on determining the total arsenic content and speciation in wild C. sinensis, often using the U.S. EPA’s long-term exposure carcinogenic and noncarcinogenic risk models (Xiao et al., 2021). To date, no study has provided direct experimental evidence of in vivo organ accumulation from wild C. sinensis or the resulting hepatotoxic and nephrotoxic effects.

This study addresses the lack of direct experimental evidence on arsenic accumulation and related toxicity in wild C. sinensis, we carried out an in vivo study in CD-1 mice. They were orally administered either the maximum clinical dose of wild C. sinensis (representing the highest arsenic exposure attainable in a single dose) and an equivalent dose of inorganic arsenic compounds, representing the worst-case toxicological scenario. We analyses of organ-specific arsenic accumulation, organ indices, serum liver/kidney function biomarkers, and hepatic/renal histopathology. Collectively, these comparative data demonstrate that wild C. sinensis causes no detectable hepatorenal toxicity in mice at the maximum clinical dose in long-term and shows substantially safer than an equivalent total arsenic dose of inorganic arsenic compounds mixture (sodium arsenate and sodium arsenite), providing reference evidence for understanding its dietary safety.

This study assessed the hepatorenal toxicity of wild Cordyceps sinensis (C. sinensis) at its maximum clinical dose and compared it with equivalent total arsenic dose of inorganic arsenic compounds mixture (sodium arsenite and sodium arsenate). The results demonstrate that administering wild C. sinensis at the maximum clinical dose does not produce significant changes in organ coefficients, tissue arsenic levels, serum biomarkers of liver function, and liver histopathological morphology in mice. Furthermore, no significant alterations were observed in serum biomarkers of renal function at weeks 2 and 4, nor in renal histopathology throughout the treatment period. In contrast, equivalent doses of inorganic arsenic compounds significantly reduced organ-to-body weight ratios, increased arsenic accumulation, impaired liver and kidney function, and induced pronounced tissue damage. Collectively, these findings indicate that under the tested conditions, wild C. sinensis does not elicit hepatorenal toxicity comparable to that of inorganic arsenic compounds, providing novel evidence supporting its safety.

At the systemic level, the stable body weight and organ-to-body weight ratios observed in the C. sinensis treatment group underscore its minimal toxicity. In contrast, inorganic arsenic exposure led to significant decreases in both body weight and organ-to-body weight ratios, particularly affecting liver and kidney mass. This finding aligns with Basher et al., who reported reduced kidney and heart weights following prenatal arsenic exposure (Basher et al., 2023), and with Lai et al., who reported significant decreases in body weight and liver mass after chronic inorganic arsenic administration (Lai et al., 2024). The reduction in organ mass likely stems from oxidative stress-induced cellular damage. Hughes and Xie et al. demonstrated that inorganic arsenic exacerbates reactive oxygen species (ROS) production, leading to excess free radical accumulation. This oxidative process damages cell membranes, proteins, and DNA and ultimately triggers apoptosis and necrosis, reducing the cellular density and organ mass (Hughes, 2002; Xie et al., 2004). Thus, wild C. sinensis showed no systemic toxicity under the tested conditions, whereas inorganic arsenic-induced toxicity clearly correlated with tissue-specific arsenic accumulation.

The quantification of arsenic levels in target organs further validated the pivotal role of tissue-specific arsenic accumulation in mediating toxicity. The Cordyceps group presented undetectable arsenic levels in liver and kidney tissues, indicating minimal arsenic burden. In contrast, the arsenic group presented substantial arsenic accumulation, supporting the hypothesis that tissue accumulation determines arsenic toxicity (Naujokas et al., 2013). Our findings align with those of Xie et al. and Flora et al., who reported dose-dependent arsenic accumulation in hepatic and renal tissues (Flora et al., 1997; Xie et al., 2004). Therefore, arsenic-induced hepatorenal toxicity depends on tissue accumulation, whereas the extremely low arsenic levels observed in the Cordyceps group suggest that under the tested conditions, the accumulation risk is negligible.

Furthermore, comparing the dosing employed in this study with international regulatory standards enhances the toxicological relevance of our findings. According to the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) guideline Q3D, the permissible daily exposure (PDE) for arsenic is set at 15 μg/day, with a No-Observed-Adverse-Effect Level (NOAEL) of 0.3 μg/kg/day (ICH, 2022). Similarly, the U.S. Environmental Protection Agency (EPA) has set an oral Reference Dose (RfD) for inorganic arsenic at 0.3 μg/kg/day (EPA, 2017), and the Agency for Toxic Substances and Disease Registry (ATSDR) defines the Minimal Risk Level (MRL) for intermediate (15–364 days) and long-term (≥365 days) inorganic arsenic exposure at the same level (0.3 μg/kg/day) (ATSDR, 2007), which is equivalent to a mouse dose of 3.69 μg/kg/day. In this study, the total arsenic content of wild C. sinensis was 16.36 mg/kg, corresponding to an equivalent murine dose of 0.03 mg/kg. This exceeds internationally established limits and is therefore predicted to lead to arsenic accumulation in mice. Nevertheless, 4-week organ--specific analysis revealed no significant increase in arsenic concentrations in either liver or kidney tissues in the Cordyceps group. In contrast, mice receiving the same dose of inorganic arsenic accumulated 26 ± 5 ng/g in the liver and 38 ± 4 ng/g in the kidneys. Xi et al. demonstrated that hepatic arsenic burdens remain below 100 ng/g under threshold exposure conditions (Xi et al., 2010), and Jomova et al. similarly reported minimal organ accumulation in populations complying with regulatory standards (Jomova et al., 2015). In summary, these data further underscore the favorable safety profile of wild C. sinensis under the tested conditions, as no arsenic accumulation in organs was observed even at exposure levels exceeding international standards.

Further analyses, including serum biochemical markers and histopathological examinations, clearly revealed the toxicological differences between wild C. sinensis and equivalent doses of inorganic arsenic. The administration of wild C. sinensis resulted in no significant hepatic or renal injury, whereas inorganic arsenic exposure significantly increased the serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, which was accompanied by liver pathology characterized by hepatocellular necrosis, steatosis, and inflammatory infiltration. These outcomes are consistent with those of Flora and Liu et al., who reported that oxidative stress and mitochondrial dysfunction are critical arsenic-induced hepatotoxic mechanisms (Flora et al., 1997; Liu and Waalkes, 2008). Additionally, the arsenic in wild C. sinensis may be metabolized in the liver through arsenic methyltransferase (As3MT). This mechanism is consistent with findings from studies on the Realgar-Indigo naturalis formula (RIF), which modulates the activity of CYP450 enzymes and the expression of As3MT, influencing arsenic methylation metabolism and thereby reducing its toxicity (Jiang et al., 2023; Xu et al., 2017). Renal toxicity is manifested by elevated blood urea nitrogen (BUN) and creatinine (CRE) levels, as well as histopathological damage to the renal tubules, which was observed only in the mice treated with inorganic arsenic, corroborating previously described oxidative stress-mediated renal toxicity (Flora, 2011; Liu et al., 2001; Xu et al., 2017). Collectively, these results demonstrate that under the tested conditions, the hepatorenal toxicity of wild C. sinensis is superior to that of equivalent doses of inorganic arsenic. This effect may be attributable to the hepatoprotective and nephroprotective activities of bioactive constituents in wild C. sinensis, such as polysaccharides and H1A-type sterols (Lin and Li, 2011).

Despite these promising outcomes, our study is limited by its 4-week duration, which reflects only medium-term toxicological responses. Long-term safety data and chronic exposure studies of wild C. sinensis are necessary. Additionally, our control group comprised inorganic arsenic compounds with equivalent arsenic contents but greater intrinsic toxicity, potentially limiting direct comparisons. Further speciation analyses of arsenic in wild C. sinensis are warranted to elucidate the distinct biological roles and toxicities of its organic arsenicals. Moreover, subsequent experiments will consider adding a male control group and increasing the sample size to enhance the generalizability of the data. Finally, while our data suggest that oxidative stress is a plausible mechanism underlying arsenic-induced hepatorenal injury, this study did not directly assess oxidative markers, and further mechanistic studies are needed for a comprehensive understanding.

In this study, we assessed the relative safety of wild Cordyceps sinensis (C. sinensis) by comparing its hepatorenal toxicity with that of a mixture of inorganic arsenic compounds (sodium arsenate and sodium arsenite) administered at an equivalent total arsenic dose. Despite containing inorganic arsenic, wild C. sinensis did not induce significant arsenic accumulation in major organs, including the heart, liver, spleen, lung, kidney, and brain, nor did it cause notable hepatorenal toxicity at its maximum clinical dose in long-term. In contrast, the inorganic arsenic compounds mixture led to marked arsenic accumulation in the liver and kidney, accompanied by evident organ dysfunction. These findings demonstrate that wild C. sinensis, when used at its clinically recommended dose, exhibits substantially lower toxicity and tissue arsenic burden compared to equivalent dose of inorganic arsenic compounds mixture, providing important evidence to support its relative safety.