A Study of Carry-Over and Histopathological Effects after Chronic Dietary Intake of Citrinin in Pigs, Broiler Chickens and Laying Hens

Abstract

Citrinin (CIT) is a polyketide mycotoxin occurring in a variety of food and feedstuff, among which cereal grains are the most important contaminated source. Pigs and poultry are important livestock animals frequently exposed to mycotoxins, including CIT. Concerns are rising related to the toxic, and especially the potential nephrotoxic, properties of CIT. The purpose of this study was to clarify the histopathological effects on kidneys, liver, jejunum and duodenum of pigs, broiler chickens and laying hens receiving CIT contaminated feed. During 3 weeks, pigs (n = 16) were exposed to feed containing 1 mg CIT/kg feed or to control feed (n = 4), while 2 groups of broiler chickens and laying hens (n = 8 per group) received 0.1 mg CIT/kg feed (lower dose group) and 3 or 3.5 mg CIT/kg feed (higher dose group), respectively, or control feed (n = 4). CIT concentrations were quantified in plasma, kidneys, liver, muscle and eggs using a validated ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method. Kidneys, liver, duodenum and jejunum were evaluated histologically using light microscopy, while the kidneys were further examined using transmission electron microscopy (TEM). Histopathology did not reveal major abnormalities at the given contamination levels. However, a significant increase of swollen and degenerated mitochondria in renal cortical cells from all test groups were observed (p < 0.05). These observations could be related to oxidative stress, which is the major mechanism of CIT toxicity. Residues of CIT were detected in all collected tissues, except for muscle and egg white from layers in the lowest dose group, and egg white from layers in the highest dose group. CIT concentrations in plasma ranged between 0.1 (laying hens in lower dose group) and 20.8 ng/mL (pigs). In tissues, CIT concentrations ranged from 0.6 (muscle) to 20.3 µg/kg (liver) in pigs, while concentrations in chickens ranged from 0.1 (muscle) to 70.2 µg/kg (liver). Carry-over ratios from feed to edible tissues were between 0.1 and 2% in pigs, and between 0.1 and 6.9% in chickens, suggesting a low contribution of pig and poultry tissue-derived products towards the total dietary CIT intake for humans.

Keywords: broiler chickens; carry-over; chronic dietary intake; citrinin; depletion; laying hens; pigs; toxicity.

Figure 1

CIT concentrations in plasma at the start of the feeding trial (day 0) until day 21 in pigs (n = 16). Day 22–25 (arrow) represents concentrations after administration of control feed, hence representing the depletion of CIT (n = 2 per day).

Figure 2

Citrinin (CIT) plasma concentrations at the start of the feeding trial (day 0) until day 21 in broiler chickens and laying hens (n = 8 per dose and species). Eight layers received CIT contaminated feed at 0.1 mg/kg, while the other 8 animals received CIT contaminated feed in a concentration of 3.5 mg/kg. Day 22–24 (arrow) represents concentrations after administration of control feed, hence representing the depletion of CIT (n = 2 per day, species and dose).

Figure 3

Cortical renal cells from the proximal convoluted tubules from (A) control pig; (B) pig in the experimental group exposed to 1 mg citrinin (CIT)/kg feed during 21 days; (C) pig in the experimental group 3 days after cessation of exposure to 1 mg CIT/kg feed during 21 days. Degenerated mitochondria are present in (B) (black arrows). Note a large vacuole with digested material (white arrow). Degenerated mitochondria are still present in (C) to a lesser extent. Magnification 2500.×. Scale bars: 2 µm.

Figure 4

Cortical renal cells from proximal convoluted tubules from (A) control broiler chicken; (B) broiler chicken in the experimental group exposed to 0.1 mg citrinin (CIT)/kg feed during 21 days; (C) broiler chicken in the experimental group exposed to 3 mg CIT/kg feed during 21 days; (D) control laying hen; (E) laying hen in the experimental group exposed to 0.1 mg CIT/kg feed during 21 days; (F) laying hen in the experimental group exposed to 3.5 mg CIT/kg feed during 21 days. Degenerated mitochondria are present in all treated animals (arrows); note remaining cristae inside the vesicles (arrowhead). The stars indicate examples of degenerating mitochondria, with increased space in between the cristae. Magnification: 2500×. Scale bars: (A,D,E):2 µm; (B,C,F): 1 µm.

Figure 5

Magnified photograph of Figure 4B, showing clear spaces between cristae of mitochondria (stars) and mitochondrial degeneration (arrows).

Figure 6

Cortical renal cells from proximal convoluted tubules from (A) broiler chicken in the experimental group 3 days after cessation of exposure to 3 mg citrinin (CIT)/kg feed during 21 days; (B) laying hen in the experimental group 3 days after cessation of exposure to 3.5 mg CIT/kg feed during 21 days. Note degenerated mitochondria in (A), implying recovery from oxidative stress, and the high number of degenerated mitochondria in (B). Magnification: 2500×. Scale bars: 2 µm.