Two Common Food Molds, One Hidden Threat: Mycotoxin Mix Silently Damages Rat Kidneys

Two of the world’s most widespread food contaminants, aflatoxin and ochratoxin A, may be far more dangerous together than either is alone, and the worst damage may be the kind that standard blood tests fail to catch. That is the central warning from a new toxicological study published in Discover Toxicology, in which researchers in Patna, India, exposed Charles Foster rats to the two mycotoxins individually and in combination over 30, 45, and 60 days. Their results reveal a troubling disconnect: in animals receiving both toxins, kidney tissue suffered progressive, chronic damage even as the biochemical markers clinicians normally rely on returned to near-normal levels, a pattern the authors describe as a form of silent toxicity.

Mycotoxins are toxic secondary metabolites produced by fungi, and more than 400 different types have been identified from over 100 fungal strains. They contaminate agricultural products through molds such as Aspergillus, Penicillium, and Fusarium, and by some estimates roughly a quarter of the world’s crops, including cereals, pulses, oilseeds, and nuts, are affected by mold and fungal contamination. Humans and animals are exposed mainly through ingestion, though dermal and inhalation routes also matter, and the severity of harm depends on the toxin type, the dose and duration of exposure, nutritional status, and interactions with other chemicals. Because real-world diets rarely contain a single contaminant, the question of how mycotoxins interact inside the body is not academic; it is central to food safety.

Aflatoxin is among the most notorious of these compounds. Produced primarily by Aspergillus flavus, A. parasiticus, A. nomius, and A. aflatoxiformans, the aflatoxin family includes 18 known types, of which AFB1, AFB2, AFG1, AFG2, AFM1, and AFM2 are the most toxic and are classified as Group 1 carcinogens. The liver is the primary target. Aflatoxin B1 is metabolized by microsomal CYP450 enzymes into AFB1-8,9-epoxide, a highly reactive form that binds DNA and proteins and drives tissue toxicity; a characteristic G to T transversion at codon 249 of the P53 gene has been linked to aflatoxin-induced hepatocellular carcinoma. An estimated 28 percent of hepatocellular carcinoma cases worldwide are associated with AFB1 exposure, and acute aflatoxicosis outbreaks have been reported in India, Kenya, and Tanzania.

Ochratoxin A, the second toxin in the study, is produced by filamentous fungi including Aspergillus and Penicillium species and exists in three forms, with OTA being the most toxic and the most frequently detected in food and feed. It exerts a remarkably broad toxicological portfolio: mutagenicity, hepatotoxicity, neurotoxicity, nephrotoxicity, teratogenicity, and immunotoxicity. OTA has been implicated as a possible etiological factor in Balkan Endemic Nephropathy, a mysterious tubulo-interstitial kidney disease seen in the Balkan Peninsula and Romania, and has been associated with chronic interstitial nephropathy in humans as well as renal and urothelial cancers in rats. Unlike aflatoxin, OTA is considered a cumulative toxin, building up in the body through repeated dietary exposure.

To test what happens when these two contaminants meet, the team, led by Kanchan Gopal Choudhary of Patna University together with colleagues at Mahavir Cancer Sansthan and Research Centre, ran a carefully controlled sub-acute toxicity experiment. Adult male Charles Foster rats, roughly eight weeks old and weighing 160 to 180 grams, were randomly assigned to groups receiving aflatoxin at 0.9 milligrams per kilogram of body weight per day, ochratoxin A at 2.75 milligrams per kilogram per day, or a combined half-dose regimen of 0.45 plus 1.375 milligrams per kilogram per day, with untreated controls for comparison. Each treatment arm was subdivided into 30-, 45-, and 60-day exposure periods. The toxins themselves were extracted from toxigenic fungal cultures, aflatoxin from Aspergillus flavus isolated from contaminated stored wheat and ochratoxin A from Aspergillus niger isolated from contaminated sesame seeds, and their concentrations were verified by thin-layer chromatography and high-performance liquid chromatography with fluorescence detection.

The hematological findings pointed to anemia across the single-toxin groups. Aflatoxin-treated rats showed significant drops in red blood cell counts, hemoglobin, platelets, lymphocyte percentage, basophil counts, and packed cell volume, alongside rises in neutrophil percentage, mean corpuscular volume, and mean corpuscular hemoglobin, a pattern consistent with normocytic normochromic anemia. The ochratoxin A group displayed a similar but distinct signature, with reduced red cells, platelets, packed cell volume, lymphocytes, monocytes, and basophils and elevated MCV, MCH, and MCHC, suggesting normocytic hypochromic anemia. The authors attribute these declines to mechanisms including hematopoietic cellular abnormalities, reduced iron-binding capacity, and inhibition of protein synthesis through decreased serum albumin. Notably, the combined-exposure group produced no clear hematological pattern at all, an early hint that co-exposure behaves in unpredictable ways.

Liver chemistry told a story of accumulating hepatic stress. Serum AST, a transaminase released when hepatocytes die, rose significantly in the 60-day groups of all three treatment arms, while alkaline phosphatase climbed significantly in the aflatoxin and ochratoxin A groups. Other liver markers, including ALT, total bilirubin, and direct bilirubin, remained unchanged, indicating that prolonged rather than short-term exposure is what tips the liver into measurable dysfunction. Under the microscope, the damage tracked the biochemistry: aflatoxin-treated livers progressed from cytoplasmic vacuolization and sinusoidal dilation at 30 days to focal necrosis at 45 days and, by 60 days, degenerated cytoplasm, irregular Kupffer cell nuclei, and mild hepatic steatosis. Ochratoxin A and the combination produced comparable trajectories of ballooning hepatocytes, periportal hemorrhage, mononuclear infiltration, and necrosis, with quantitative scoring of degeneration among 100 hepatic cells per animal confirming steadily worsening injury over time.

The kidney results were where the study took its most unsettling turn. In aflatoxin-treated rats, creatinine, a standard marker of renal filtration failure, rose significantly at 30 and 45 days but returned to near-control values by 60 days, a rebound the authors suggest may reflect induction of cytochrome P-450 enzymes such as CYP3A4 that metabolize aflatoxin with prolonged exposure. Ochratoxin A produced the expected nephrotoxic profile, with uric acid elevated at 45 days and creatinine elevated at both 45 and 60 days, confirming dose- and time-dependent renal injury. Histologically, all treated groups showed glomerular degeneration, damage to the brush border of Bowman’s capsule, cytoplasmic vacuolization of the epithelial lining, and degeneration of proximal and distal convoluted tubules, with hemorrhages inside Bowman’s capsule appearing in the combined group by 60 days.

The combination group, however, broke the expected correlation between blood chemistry and tissue damage. Creatinine rose significantly only at 45 days and had normalized by 60 days, while uric acid, urea, and blood urea nitrogen never significantly changed, yet histopathological analysis of the same animals revealed chronic, progressive kidney toxicity, including tubular destruction, glomerular degeneration, and hemorrhage. In other words, the laboratory values suggested recovery or tolerance at precisely the time point when the tissue showed the most severe structural damage. The authors conclude that aflatoxin plus ochratoxin A may act as a silent toxicant, one that evades the standard biomarker panel used to detect kidney injury, and they caution that biochemical normality should not be mistaken for histological health in co-exposure scenarios.

The findings carry real-world weight because co-contamination of food and feed with multiple mycotoxins is the norm rather than the exception, and regulatory limits are typically set toxin by toxin. If half-doses of two common contaminants can quietly destroy renal tissue while leaving blood tests clean, current monitoring strategies may systematically underestimate the health burden of mycotoxin mixtures, particularly in regions where dietary exposure is chronic and healthcare diagnostics rely heavily on serum chemistry. The authors acknowledge a key limitation: toxin levels in the blood were not measured, so pharmacokinetic interactions between aflatoxin and ochratoxin A remain to be characterized. Follow-up studies tracking co-exposure with direct toxin quantification, and ideally with more sensitive kidney injury biomarkers beyond creatinine, will be needed to confirm how generalizable this silent-toxicity phenomenon is, and whether human populations eating contaminated staples face the same invisible risk.

Subject of Research: Sub-acute toxicity of combined aflatoxin and ochratoxin A exposure in Charles Foster rats

Article Title: Synergistic impact of aflatoxin and ochratoxin A exposure in Charles foster rats: a toxicological study

Article References: Choudhary, K. G., Sharfuddin, C., Kumar, A., & Ghosh, A. K. (2025). Synergistic impact of aflatoxin and ochratoxin A exposure in Charles foster rats: a toxicological study. Discover Toxicology, 2(1), Article 16. https://doi.org/10.1007/s44339-025-00036-8

Image Credits: AI Generated

DOI: 10.1007/s44339-025-00036-8

Keywords: aflatoxin, ochratoxin A, mycotoxins, nephrotoxicity, hepatotoxicity, silent toxicity, Charles Foster rats, food safety, normocytic anemia, histopathology, creatinine, toxicology