Placental and lactational transfer of ochratoxin A in rats.

The placental and lactational transfer of ochratoxin A (OA) was investigated in a cross-fostering study in rats. Dams were given 50 microg OA(-1) kg body weight by gastric intubations 5 times a week for 2 weeks before mating, during gestation and then 7 days a week during lactation. Neonates from OA-treated dams were cross-fostered at birth to control dams treated with only vehicle. In the same way, neonates from control dams were cross-fostered to OA-treated dams. Treatment with OA did not result in any effects on birth weight or growth development of the pups during the first 21 days of life. There were no effects on milk quality as measured by milk lipids, protein or lactose concentrations, or on milk production, assessed by the mammary gland content of RNA and DNA. A mean milk:blood ratio of approximately 0.6 was found. The dose of OA from milk to the suckling pup at 14 days of age can be calculated to about 50 microg kg(-1) body weight(-1) day, which is similar to the dose given to the dams. Pups exposed to OA only via milk had blood and kidney levels of OA approximately 3 times higher than their dams, indicating a high absorption and/or a low excretion of OA in the sucklings. At 14 days of age the highest blood and kidney levels of OA were found in offspring exposed both via placenta and milk, with the highest contribution from milk. Offspring exposed only via milk had about 4-5 times higher levels of OA in blood and kidney compared to offspring exposed only via placenta. As milk could be a significant source of OA exposure in newborns, adverse health effects resulting from postnatal exposure should be studied and evaluated in the risk assessment of OA.

Prenatal exposure of Balb/c mice to ochratoxin A: effects on the immune system in the offspring.

Effects of prenatal exposure to the mycotoxin ochratoxin A (OA) on the immune system were evaluated in Balb/c mice. Dams were exposed to OA in their diet at doses of 0.18 (control), 30 or 200 micrograms/kg before and during gestation. At birth, pups were cross-fostered to non-exposed dams. OA exposure of the dams did not influence reproductive outcome, that is, the numbers of litters, litter sizes and body weight of the pups. Flow cytomety analysis of T-lymphocyte subpopulations on days 14 and 28 postpartum revealed a decrease in the percentages of splenic CD4+ and CD8+ cells in offspring from the high-dose group (200 micrograms/kg diet), but no significant alterations in absolute numbers of these cell populations nor in the total numbers of splenocytes were observed. In the thymus, a relative as well as an absolute increase in the CD4+ subpopulation was seen in exposed pups on day 14. On day 28, the absolute numbers of CD4+, CD8+ and CD4+CD8+ (double positive) cells were increased, reflecting an elevated number of thymocytes in the high-dose group. No significant differences were found in the proliferative responses of splenic or thymic lymphocytes to mitogens, or in the production of interleukin-2 in concanavalin A-stimulated cell cultures. Further, the plaque-forming cell response to sheep red blood cells and the humoral antibody response to the viral antigen PR8 were not affected by prenatal exposure to OA. No significant differences in natural killer cell activity were observed. The results indicate that exposure of dams to relatively low levels of dietary OA alters absolute and relative numbers of lymphocyte subpopulations in lymphoid organs, but does not suppress immune functions in the offspring.

Influence of perinatal ochratoxin A exposure on the immune system in mice.

The mycotoxin ochratoxin A (OA) is a well-documented immunotoxic agent which affects both cellular and humoral immunity. In the present study, the effects of maternal exposure to single doses of OA during gestation or lactation were studied in Balb/c offspring. A single dose exposure of the dams to OA (500 micrograms/kg body weight) on day 16 of gestation resulted in decreased proliferation of splenic and thymic lymphocytes in response to mitogens in the pups at 15 days of age. Flow cytometry analysis of thymocyte subpopulations revealed lower percentages of mature CD4+ cells and higher percentages of immature, double-positive (CD4+CD8+) cells in the exposed pups. In contrast, a single exposure of the dams of OA on day 10 postpartum significantly increased the proliferative responsiveness of lymphocytes in the offspring when stimulated with B or T cell mitogens 3 days after the exposure. This effect was most prominent in the highest dose group (500 micrograms/kg body weight). The present results are in accordance with previous observations in rats, and show that the time of exposure significantly influences the immunotoxic effects of OA on the developing immune system in rodents.

Effects of ochratoxin A on the rat immune system after perinatal exposure.

Effects on the immune system after perinatal exposure to ochratoxin A (OA) were studied in Sprague-Dawley rats after single or repeated exposure of the dams. In a short-term study, dams with litters were given a single dose of OA (0, 10, 50 or 250 micrograms/kg body weight) on day 11 of lactation. The effects on cell numbers in spleen and thymus añd on the mitogen responses of lymphocytes were evaluated in the suckling pups on day 14 of lactation. The proliferative response of splenocytes to the T-cell mitogen Concanavalin A (Con A) was significantly stimulated in pups from dams given 10 or 50 micrograms OA/kg body weight as compared to control pups. In addition, proliferation of thymocytes in response to Con A was stimulated in pups from dams exposed to 50 micrograms OA/kg body weight. In a long-term, cross-fostering study comparing pre- and postnatal exposure, half of the dams received 50 micrograms OA/kg body weight 5 days a week by gastric intubation 2 weeks before mating, during gestation and then 7 days a week until weaning. Effects on immune parameters were studied in the pups on day 14 of lactation and at 13 weeks of age. Suppressed mitogenic responses were seen to both lipopolysaccharide (LPS) and Con A in prenatally exposed pups sampled on day 14 of lactation. At 13 weeks the response of splenocytes to LPS was still impaired. The primary antibody response to a viral antigen was also lower in the prenatally exposed pups than in the control pups. These effects on the immune response were not seen in the groups of pups exposed postnatally or both pre- and postnatally, although blood concentrations of OA were higher in these groups at the time of the first sampling. This indicates that the decrease in proliferation and antibody production resulted from prenatal modulation of the immune system. The results show that prenatal exposure to relatively low doses of OA may induce immunosuppression. In contrast, short-term exposure of suckling pups to OA via the milk stimulates the proliferative responses of lymphocytes to polyclonal activation.

Effects of ochratoxin A on the mouse immune system after subchronic exposure.

The effects on the immune system of oral, subchronic exposure to ochratoxin A (OA) at 6, 250 or 2600 micrograms/kg diet were studied in female Balb/c mice. After 28 days of exposure, antibody production plague-forming cells/spleen, was suppressed in a dose-dependent manner which was significant in the two highest exposure groups. In addition, a decrease in thymocyte cell counts was seen in the 250-micrograms/kg group. After 90 days exposure, flow cytometry analysis of thymic lymphocyte subpopulations revealed a decreased proportion of mature (CD4+ or CD8+) cells. Furthermore, the mitogenic responsiveness of thymocytes and splenocytes to concanavalin A (Con A) was significantly decreased. This effect was observed in all three treatment groups. Interleukin-2 production of Con A-stimulated lymphocytes, natural killer cell activity, and humoral antibody titres to a viral antigen were not affected by OA treatment. The present results indicate that subchronic, oral exposure to OA affects certain immune functions in mice at exposure levels that may be found in contaminated food products.

Toxicokinetics of ochratoxin A in rat following intratracheal administration.

The toxicokinetic parameters of ochratoxin A in rat following intratracheal administration of 50 ng ochratoxin A/g body weight were studied. The absorption of ochratoxin A from the lungs was very efficient. The elimination pattern of the toxin was studied by the analysis of blood samples. The biological half-life of ochratoxin A was 127 h and the calculated apparent volume of distribution equalled 168 ml/kg. The bioavailability of the toxin was very high, 98%. The plasma clearance of the toxin was 0.92 ml/kg.h.

Ochratoxin A in human serum samples collected in southern Italy from healthy individuals and individuals suffering from different kidney disorders.

Ochratoxin A was determined in human serum samples, collected in the south of Italy in November 1992, using ion-pair liquid chromatography and fluorescence detection. The samples were collected from healthy people (65 subjects) as well as from people with different kidney disorders. Five different kinds of kidney disorders were represented: transplanted subjects (13), chronic glomerulonephritis (8), renal calculus or cyst (6), chronic renal failure (13), and subjects treated by dialysis (28). The mean and median concentrations of ochratoxin A in the healthy group was 0.53 and 0.44 ng/ml serum, respectively. The highest mean concentration was found in the group of patients treated by dialysis, 1.4 ng/ml serum. A higher incidence of samples containing > 0.44 ng ochratoxin A/ml serum was found in the dialysis group, compared to the other groups. Comparing the mean concentrations by Student's t-test, a significant difference was found between the mean concentrations of the healthy group and of the group of patients treated by dialysis (P < 0.01). No other significant differences were found when comparing the groups two at a time.

Ochratoxin A in cow's milk and in human milk with corresponding human blood samples.

A method for determining ochratoxin A in milk has been elaborated in which the sample was subjected to a liquid-liquid extraction step and then purified on a silica gel column packed in a Pasteur pipet. The purified samples were analyzed by ion-pair liquid chromatography with fluorescence detection. The detection and quantitation limits for determination of ochratoxin A in cow's milk were 10 and 40 ng ochratoxin A/L milk, respectively. The same limits were valid for the analysis of human milk. A total of 36 cow's milk and 40 human milk samples were analyzed. All samples were collected in Sweden. Ochratoxin A was found in 5 (14%) of the cow's milk samples (range 10-40 ng/mL) and in 23 (58%) of the human milk samples (range 10-40 ng/L). Blood samples were collected from the mothers who gave milk samples. A total of 39 samples were analyzed. All blood samples contained ochratoxin A in concentrations exceeding the quantitation limit (60 ng/L blood). The mean concentration of ochratoxin A in the samples was 167 ng/L blood (range 90-940 ng/L). The concentration of ochratoxin A in human milk was < or = 0.1 of that in the human blood.

Transfer of ochratoxin A from lactating rats to their offspring: a short-term study.

A dose-dependent transfer of ochratoxin A into the milk of lactating rats was found after a single oral dose of ochratoxin A, given in the dose levels of 10, 50, and 250-micrograms ochratoxin A/kg body weight by gastric intubation. The milk/blood concentration ratio of ochratoxin A at 24 and 72 h was 0.4 and 0.7, respectively. A linear relationship was found between the concentration of ochratoxin A in the dam's milk and in the blood of the pups at 72 h, as well as in the dam's milk and in the kidneys of the pups. The pup blood/milk concentration ratio of ochratoxin A was approximately 6. At 72 h the sucklings had higher levels of ochratoxin A than their dams in both blood and kidneys. The results show that the concentration of ochratoxin A in milk can be used as an indicator of the continuously administered dose to the suckling.

Synthesis of 14C-ochratoxin A and 14C-ochratoxin B and a comparative study of their distribution in rats using whole body autoradiography.

Methods for preparation of labelled ochratoxin A and B are described. The method for preparation of labelled ochratoxin B involves the synthesis of the azide of ochratoxin beta via the mixed anhydride and subsequent conjugation to labelled phenylalanine to yield 14C-ochratoxin B. The labelled ochratoxins were injected into male Wistar rats and after different survival times they were sacrificed and subjected to whole body autoradiography. The distribution pattern of ochratoxin A in the rat did not differ from that earlier registered for mouse. The previously known, high susceptibility of rats (and not mice) to ochratoxin A-induced cancer could thus not be explained by an accumulation of the toxin in specific cells or organs. The distribution patterns of ochratoxin A and B were almost congruent--the only apparent difference being a much longer retention of the labelled ochratoxin A in the blood compared to ochratoxin B, which was much faster excreted. When analyzing tissue extracts for labelled metabolites only the extracts from the rats injected with ochratoxin B were found to contain easily detectable concentrations, while no metabolites of ochratoxin A were seen.

Penicillium verrucosum in feed of ochratoxin A positive swine herds.

Ochratoxin A contamination of cereal feed grain was monitored during October 1989-September 1990 by analysis of blood samples from slaughter swine in Sweden. The detection of ochratoxin A in swine blood was used as a method to identify swine herds fed ochratoxin A contaminated feed. The contamination level of ochratoxin A in the blood of the positive herds was in the range 2-45 ng/ml with the mean concentration 5.2 ng/ml. Feed samples for mycological analysis were collected from both ochratoxin A positive herds (greater than or equal to ng/ml blood) and ochratoxin A negative herds (less than 2 ng/ml blood). From the ochratoxin A positive herds and the ochratoxin A negative herds 22 and 21 feed samples were collected, respectively. No quantitative differences in mould content, as determined by colony forming units, were observed between the two groups. However, there were differences in the mycoflora. The incidence of storage fungi (Penicillium and Aspergillus spp.) was significantly higher (p less than 0.05) in feed from ochratoxin A positive herds. Particularly, Penicillium verrucosum was found to be significantly more common (p less than 0.001). Altogether 274 isolates were screened for their ability to produce ochratoxin A. Ochratoxin A producers were found only within P. verrucosum; 38% of the 63 isolates produced detectable amounts of ochratoxin A. Ochratoxin A producing isolates of P. verrucosum were found in 60% of the feed samples collected from ochratoxin A positive swine herds and in one sample (5%) of the feed samples collected from the ochratoxin A negative herds.

Distribution of 14C-ochratoxin A and 14C-ochratoxin B in rats: a comparison based on whole-body autoradiography.

The distribution patterns of ochratoxin A and its nontoxic dechloro-analogue ochratoxin B were studied in rats using whole-body autoradiography. No prominent difference in distribution patterns was found that could explain why the rat is the animal most susceptible to ochratoxin A-induced renal cancer or why ochratoxin B is less toxic than ochratoxin A.

Enzyme-linked immunosorbent assay for analysis of ochratoxin A in human plasma samples using antibodies raised against a new ochratoxin-protein conjugate.

A derivative of ochratoxin A was linked to bovine serum albumin in such a way that the carboxylic group of the toxin was left free. Injection of the conjugate into rabbits resulted in sensitive antibodies towards ochratoxin A, which were used in an enzyme-linked immunosorbent assay.