Leonard Friedman

In vitro effects of the nephrotoxins ochratoxin A and citrinin upon biochemical function of porcine kidney

Ochratoxin A and citrinin are nephrotoxic mycotoxins found in a variety of foods and feeds. Before studying possible interactions between these two toxins, their individual biochemical effects were examined in vitro by using renal cortical explants derived from male swine of the Hormel-Hanford strain. The following measurements were performed: macromolecule biosynthesis (protein, RNA, and DNA), respiration (14CO2 from [14C]glucose), organic ion (tetraethyl ammonium acetate, i.e., TEA) transport, and membrane perturbation (protein leakage into medium). Levels of the toxins ranged from 0.001 to 1 mM. Ochratoxin A inhibited macromolecule biosynthesis at a lower concentration (0.001 mM) than did citrinin. Protein and DNA synthesis were particularly sensitive to ochratoxin A. Syntheses of protein and DNA were inhibited at ochratoxin A concentrations of 0.01 and 0.001 mM, respectively. RNA synthesis was less sensitive to the mycotoxin; it was inhibited only 60% at 1 mM, the highest concentration of ochratoxin A tested. Citrinin levels of 0.01 mM were required for inhibition of RNA, DNA, and protein synthesis. Inhibition by citrinin was approximately equal for all three classes of macromolecules. Citrinin was more effective than ochratoxin A in the inhibition of respiration and TEA transport; the minimum effective levels of citrinin were 1 and 0.01 mM, respectively. Serious membrane damage as evidenced by increased protein leakage was not caused by either toxin. Stimulation of respiration, perhaps reflective of uncoupling of oxidative phosphorylation, was produced by an ochratoxin A concentration of 1 mM. Inhibition of macromolecule biosynthesis does not seem to be related to the effect of either toxin upon respiration, although a causal relationship between the effects of ochratoxin A on respiration and TEA transport cannot be ruled out.

Kinetic analysis of glutamate transport by the miniswine choroid plexus in vitro

Transport of glutamic acid by the choroid plexus, the blood-cerebrospinal fluid (CSF) barrier, was investigated by using the isolated choroid plexi from the fourth (FVCP) and lateral ventricles (LVCP) of the young adult miniswine in vitro. Glutamic acid uptake was very pronounced, with concentrations 7-fold (LVCP) and 2.4-fold (FVCP) higher in tissue than in medium after only 5 min of incubation with 1 microM glutamic acid. Tissue/medium ratios reached steady state by 15 min at 30-fold (LVCP) and 11-fold (FVCP). Uptake was energy-dependent and inhibited by ouabain and hypothermia. L-Aspartic acid was shown to be inhibitory in a concentration-dependent manner, suggesting that it shares a common transport system, whereas neither octanoic acid nor okadaic acid (transported by a separate fatty acid system) inhibited glutamic acid transport. At the same temperature, the labeled metabolite of glutamate (glutamine) in the tissue was 64.7%, 73.2%, and 72.5% of total radioactivity at 5, 30, and 60 min, respectively. The estimated Km values for glutamate uptake by the choroid plexus are 264 microM (FVCP) and 196 microM (LVCP); Vmax values are 87 (FVCP) and 147 (LVCP) nmol/g/min, respectively. These results indicate that, in addition to the metabolism of glutamate to glutamine, an active uptake mechanism is present in the choroid plexus of miniswine which may serve to regulate glutamic acid concentration in the CSF.

BODY AND ORGAN GROWTH OF THE DEVELOPING HORMEL-HANFORD STRAIN OF MALE MINIATURE SWINE

As part of a larger study designed to characterize the early developmental stages of the Hormel-Hanford strain miniature pig, whole body, brain, kidney, liver, pancreas and spleen from male animals were examined for weight increases from one to 196 days, the approximate age of maturity. At 196 days, body weights had increased to 82.5 times the weight at day 1; increases in organ weights were greatest for spleen, less and similar for kidney, liver and pancreas, and the least for brain. Little change in relative organ weights was noted, except for the brain where an almost steady decrease occurred starting from 7 days after birth.

Patulin: Mycotoxin or Fungal Metabolite? (Current State of Knowledge)

Following the discovery of penicillin by Fleming in 1929 a flurry of work ensued in which similar approaches were applied to the search for other antibiotics. One of the products of this search was the compound patulin, initially isolated from filtrates of a strain of Penicillium claviforme by Chain et al. (1942). The compound, originally referred to as clariformin and possessing potent antibacterial activity, was the subject of numerous chemical and biological studies soon after its isolation from a strain of Penicillium patulum Painier and characterization by Birkinshaw et al. (1943). Elucidation of the structure was made by Woodward and Singh (1949). It has since been shown to be produced by several species of Aspergillus and Penicillium and at least one species of Byssochlamys, as reviewed by Ciegler (1977) and Stott and Bullerman (1975).

INTERACTION OF AFLATOXINS AS MEASURED BY THEIR BIOCHEMICAL ACTION ON RAT LIVER SLICES AND HEPATOCYTES

The possible interaction between pairs of aflatoxins com monly occurring together in foods was evaluated by determining their biochem ical actions on the liver and hepatocytes of young mature rats. RNA synthesis, a sensitive and 14 early target of the effects of aflatoxin B1, was m easured with [ C]orotic acid or 3 [ H]uridine used as substrates. In the first study (comprised of four replicate experiments), liver slices treated with aflatoxin B1 at 0, 120, 240, and 480 ng/ ml were incubated in the presence and absence of aflatoxin B2 at 120 ng/ ml. In the second study, liver slices were treated with the sam e concentrations of B1 incubated in the presence and absence of aflatoxin G1 at 120 ng/ m l (comprised of four replicate experim ents) or at 240 ng/ ml (comprised of two replicate experim ents). In the third study (single experim ent), isolated hepatocytes were treated with aflatoxin B1 at 0, 60, 120, 240, and 480 ng/ ml in the presence and absence of aflatoxin G1 at 120 ng/ m l. Leakage of lactic acid...

Ornithine decarboxylase and thymidine kinase activities and polyamine levels from selected organs of adult miniature swine receiving three concentrations of dietary menhaden oil

Mature, female swine were randomly assigned to one of seven dietary groups. Swine in groups 1-3 were fed a cholesterol-rich diet for 55 days while the remaining groups remained on a basal swine diet. At the end of the cholesterol(Chol)-preloading period the swine in groups 1-7 were placed on menhaden oil (MO) and/or corn oil (CO) as follows: groups 1 and 4, 15% CO (control); groups 2 and 5, 0.75% MO+14.25% CO; groups 3 and 7, 15% MO; and group 6, 7.5% MO+7.5% CO. Animals were killed at the end of the approximately 6-month feeding period and portions of liver, pancreas and colon mucosa were analyzed for both ornithine decarboxylase (ODC) and thymidine kinase (TK) activity while polyamine levels were measured in the liver and pancreas. Statistical analyses were carried out by one-way and two-way ANOVA and by trend analysis. In the pancreas, the highest MO group (group 7) had significantly higher ODC levels when compared with the CO control (group 4) and the next to highest MO group (group 6) (one-way ANOVA)-all non-cholesterol preloaded groups. Using a two-way ANOVA (Chol-by-MO), liver ODC was significantly lower in the CO control when compared with the lowest and highest MO groups (groups 5 and 7, respectively), again in the non-cholesterol-preloaded animals. In the colon, the swine in the Chol-low MO group (group 2) had significantly lower TK activity than the Chol/CO control group (group 1) and Chol/Hi MO group (group 3) (one-way ANOVA) and also had significantly lower activity than all groups except the CO control (group 4) (two-way ANOVA). Liver acetylputrescine in the lowest and highest MO groups (groups 5 and 7, respectively) was significantly higher than in the CO group (group 4). Liver spermidine in the Chol-Hi MO group (group 3) was significantly higher than the Chol-Lo MO group (group 2), while the highest MO group (group 7) had a statistically higher level than the other non-cholesterol groups (groups 4-6) (one-way ANOVA). Liver spermine was significantly higher in the Chol-Hi MO group (group 3) when compared to the CO control (group 1) and the Chol-Lo MO group (group 2) (one-way ANOVA). Pancreatic putrescine in the CO control (group 4) was significantly higher than all other groups (two-way ANOVA) while spermine from the 2 Chol-MO groups (groups 2 and 3) was higher than the Chol-CO control (group 1) (one-way ANOVA). Using trend analysis, liver TK, putrescine and spermidine increased in the non-cholesterol preloaded groups with increasing dietary MO, similar to the increase seen in ODC. Thus, of the three organs studied, only liver responded to menhaden oil with changes in both ODC itself or some of its metabolic engendered products and thymidine kinase; at least for one of the parameters, ODC, change associated with dietary MO was dependent on whether the swine were preloaded with cholesterol.

Distribution and Metabolism of Citrinin: A Review

Citrinin was first isolated by Hetherington and Raistrick in 1931 from a culture filtrate of Penicillium Thom. Other species of Penicillium as well as several species of Aspergillius also produce citrinin (Timonin and Rouatt, 1944; Krogh et al., 1970; Mislivec and Tuite, 1970; Ito et al., 1973; Pohland and Mislivec, 1976; Stoloff, 1976; Nelson et al., 1980; Mislivec, 1981). It is a potentially important mycotoxin; it had been found as a natural contaminant of peanuts (Diener, 1960), corn (Mislivec and Tuite, 1970), rice (Saito et al., 1971); wheat, rye, barley, and oats in Canada (Scott et al., 1972); barley and oats in Denmark (Krogh et al., 1973); naturally rotted apples containing Penicillium expansum (Harwig et al., 1973); and Indian groundnuts infected with A. flavus. P. citrinum and A. terreus (Subrahmanyam and Rao, 1974).

Ornithine decarboxylase, fatty acid synthetase, and lipid levels in selected organs of the postnatal developing male miniature pig

Ornithine decarboxylase (ODC) and fatty acid synthetase (FAS) activities were determined in tissues from male neonate and juvenile miniature swine (Hormel-Hanford strain) at various ages. ODC activity was measured in liver, brain, kidney, pancreas, and spleen at one day and at 1, 4, 8, 12 and between 24 and 32 weeks. Hepatic FAS activity, total lipid, triglyceride, and total cholesterol were measured at 2, 8, 16, and 32 weeks. Generally, tissue ODC activity was highest in the spleen at all ages. Three postnatal patterns of ODC activity were observed for the different organs. The mean values of FAS activity, total lipid, and cholesterol were highest at 8 weeks compared to other sampling periods.

Induction of ornithine decarboxylase in the stomach mucosa of swine with NaCl or 12-O-tetradecanoylphorbol-13-acetate

The effect of sodium chloride and 12-O-tetradecanoylphorbol-13-acetate on ornithine decarboxylase (ODC) activity in gastric mucosa of miniature swine was investigated as a model for gastric inflammation. The level of the enzyme was lower in the pylorus than in the fundic or cardiac regions of the stomach in untreated animals. Treatment with sodium chloride at 1 g/kg produced large increases in all three regions, with the greatest relative increase in the pylorus. Treatment with sodium chloride at 0.25 g/kg or the phorbol ester at 2.0 mg/pig produced significant but less dramatic increases. ODC activity in control and treated mucosal extracts was inhibited by the specific ODC inhibitor difluoromethylornithine. Most of the enzyme activity was associated with superficial and exfoliated cells that could be scraped from the mucosal surface. No increase in the inflammatory mediator leukotriene B4 was observed in the mucosal extracts. Ornithine decarboxylase appears to be a useful enzymatic marker for the regenerative events that occur after tissue damage and may correlate with the putative tumor-promoting function of sodium chloride in gastric tissues.

Macromolecular Levels, Dna Synthesis and Ornithine Decarboxylase Activity in Leg Muscles From 6-Mercaptopurine-Treated Rats

Sprague-Dawley male and female rats were treated with 6-mercaptopurine (6-MP) (2 mg/kg sc) daily from 2 to 22 days of age and killed at 7, 15, 27 and 64 days of age. At 7 and 27 days of age rats were injected with 3H thymidine for measurement of DNA synthesis. Fore- and hindlimb muscles were removed and analyzed for ornithine decarboxylase (ODC) activity (all ages), DNA radioactivity (7 and 27 days), DNA level (27 and 64 days) and RNA level (64 days). As expected, ODC activity and DNA synthesis were higher in muscles of 7-day-old rats than in muscles of the older rats studied. A consistently lower ODC activity was seen in 6-MP-treated vs. control rats for 5-25 days after start of treatment, but the effect was essentially the same for the hindlimb and forelimb muscles. During the 7-27-day time course ODC activity was higher in hindlimb than forelimb muscles. By 27 days of age DNA synthesis was also higher in the hindlimb muscles. DNA synthesis was decreased after 5 days of treatment relative to that of control rats, to an approximately equal extent in forelimb and hindlimb muscles. Five days after the last treatment a trend was seen for slower recovery from inhibition of DNA synthesis in hindlimb muscles, particularly in male rats. DNA levels were reduced in treated rats relative to those in control rats 5 days after the last treatment to approximately the same degree in forelimb and hindlimb muscles. Forty-two days after the last treatment a trend toward increased activity of ODC and increased DNA and RNA levels was seen in muscles of treated rats, probably reflective of recovery processes. These early biochemical effects of 6-MP, which were seen to about the same extent in the forelimb and hindlimb muscles cannot explain by themselves the delayed hindlimb fat atrophy resulting from 6-MP treatment of neonatal rats.