Climate change impacts on natural toxins in food production systems, exemplified by deoxynivalenol in wheat and diarrhetic shellfish toxins.

Climate change is expected to affect food and feed safety, including the occurrence of natural toxins in primary crop and seafood production; however, to date, quantitative estimates are scarce. This study aimed to estimate the impact of climate change effects on mycotoxin contamination of cereal grains cultivated in the terrestrial area of north west Europe, and on the frequency of harmful algal blooms and contamination of shellfish with marine biotoxins in the North Sea coastal zone. The study focused on contamination of wheat with deoxynivalenol, and on abundance of Dinophysis spp. and the possible relationship with diarrhetic shellfish toxins. The study used currently available data and models. Global and regional climate models were combined with models of crop phenology, mycotoxin prediction models, hydrodynamic models and ecological models, with the output of one model being used as input for the other. In addition, statistical data analyses using existing national datasets from the study area were performed to obtain information on the relationships between Dinophysis spp. cell counts and contamination of shellfish with diarrhetic shellfish toxins as well as on frequency of cereal cropping. In this paper, a summary of the study is presented, and overall conclusions and recommendations are given. Climate change projections for the years 2031-2050 were used as the starting point of the analyses relative to a preceding 20-year baseline period from which the climate change signal was calculated. Results showed that, in general, climate change effects lead to advanced flowering and harvest of wheat, and increased risk of contamination of wheat with deoxynivalenol. Blooms of dinoflagellates were estimated to occur more often. If the group of Dinophysis spp. behaves similarly to other flagellates in the future then frequency of harmful algal blooms of Dinophysis spp. may also increase, but consequences for contamination of shellfish with diarrhetic shellfish toxins are uncertain. Climate change will also have indirect effects on toxin contamination, which may be equally important. For example, the frequency of cropping of wheat and maize in north Europe was projected to increase under climate change, which will also increase the risk of contamination of the grains with deoxynivalenol. Risk managers are encouraged to consider the entire range of the predictions of climate change effects on food safety hazards, rather than median or average values only. Furthermore, it is recommended to closely monitor levels of mycotoxins and marine biotoxins in the future, in particular related to risky situations associated with favourable climatic conditions for toxin producing organisms. In particular, it is important to pay attention to the continuity of collecting the right data, and the availability and accessibility of databases. On a European level, it is important to stress the need for harmonisation of terminology and data collection.

A novel source for dioxins present in recycled fat from gelatin production.

Within a survey on dioxins in animal fat used as feed ingredient, a sample originating from pigs offal was shown to contain 50 ng Toxic Equivalents (TEQ) PCDD/PCDFs kg(-1) fat. Further investigation revealed fat samples with levels as high as 440 ng TEQ kg(-1) fat and contaminated feed with a highest level of 8.4 ng TEQ kg(-1) feed. The congener pattern was dominated by 1,2,3,7,8-PeCDD and 2,3,7,8-TCDD, and was not recognized from any previous incident or known dioxin source. Remarkably, 2,3,7,8-substituted congeners were much more abundant than their non-2,3,7,8-substituted counterparts. The sampled fat was derived from a gelatin production plant. Broken filters, used to clean the hydrochloric acid (HCl) used in the process, caused the dioxin contamination. The fat was primarily used for pig feed. A new physiologically-based pharmacokinetic (PBPK) model for lipophilic contaminants in growing slaughter pigs predicted levels at slaughter varying between 40 pg TEQ g(-1) fat (worst-case) and 2.5-7pgTEQ g(-1) fat under more realistic scenarios. Almost 300 farms were temporarily blocked. Many fat samples of pigs were analyzed using a combined approach of DR CALUX and GC/HRMS. Levels in contaminated pig fat were around the EU-limit of 1 pg TEQ g(-1) fat, with some samples up to 2-3 pg TEQ g(-1) fat. Of 80 negative samples analyzed by DR CALUX and GC/HRMS no false-negatives were obtained, whereas 36 and 62 of the 80 samples classified suspected with the bioassay had GC/HRMS levels above respectively the tolerance and action limits. It is concluded that novel and unexpected dioxin sources remain a threat to the food chain and require the proper evaluation and monitoring of production processes, including chemicals used therein.

Pharmacokinetics of sulfadimethoxine and sulfamethoxazole in combination with trimethoprim after oral single- and multiple-dose administration to healthy pigs.

The pharmacokinetics were studied of sulfadimethoxine (SDM) or sulfamethoxazole (SMX) in combination with trimethoprim (TMP) administered as a single oral dose (25 mg + 5 mg per kg body weight) to two groups of 6 healthy pigs. The elimination half-lives of SMX and TMP were quite similar (2-3 h); SDM had a relatively long half-life of 13 h. Both sulfonamides (S) were exclusively metabolized to N4-acetyl derivatives but to different extents. The main metabolic pathway for TMP was O-demethylation and subsequent conjugation. In addition, the plasma concentrations of these drugs and their main metabolites after medication with different in-feed concentrations were determined. The drug (S:TMP) concentrations in the feed were 250:50, 500:100, and 1000:200 mg per kg. Steady-state concentrations were achieved within 48 h of feed medication, twice daily (SDM+TMP) or three times a day (SMX+TMP). Protein binding of SDM and its metabolite was high (>93%), whereas SMX, TMP and their metabolites showed moderate binding (48-75%). Feed medication with 500 ppm sulfonamide combined with 100 ppm TMP provided minimum steady-state plasma concentrations (C(ss,min)) higher than the concentration required for inhibition of the growth of 90% of Actinobacillus pleuropneumoniae strains (n = 20).

Prevention of pleuropneumonia in pigs by in-feed medication with sulphadimethoxine and sulphamethoxazole in combination with trimethoprim.

The prophylactic effect of in-feed medication of conventional pigs with sulphadimethoxine (SDM), sulphamethoxazole (SMX), and trimethoprim (TMP) was tested by using an Actinobacillus pleuropneumoniae infection model. In each of five experiments, six pigs were given medicated feed twice daily and three pigs received antibiotic-free feed and served as positive (unmedicated, infected) controls. The following drugs or drug combinations were tested (in mg per kg feed): 500 SDM + 100 TMP, 500 SMX + 100 TMP, 125 SMX + 25 TMP, 125 SMX (alone) and 25 TMP (alone). After six days of feed medication, all animals were endobronchially inoculated with A. pleuropneumoniae in a dose of 1-3.10(4) colony-forming units (CFU). The response to the challenge in all control pigs was characterized by fever, lethargy, anorexia, reduced water consumption, and laboured breathing. At autopsy all controls manifested a fibrinous haemorrhagic pleuropneumonia. In-feed medication with 500 SDM + 100 TMP, 500 SMX + 100 TMP as well as 125 SMX + 25 TMP resulted in an effective protection against the challenge in all treated animals. After consumption of feed medicated with 125 mg per kg SMX or 25 mg per kg TMP, pleuropneumonia was evident in all challenged pigs. The results of this study indicate an in vivo potentiation of SMX and TMP in pigs against this respiratory tract pathogen.

Bioavailability of genistein, daidzein, and their glycosides in intestinal epithelial Caco-2 cells.

In this study information was obtained on bioavailability of genistein, daidzein and their glycosides in human intestinal epithelial Caco-2 cells grown on semi-permeable filters. The integrity of Caco-2 monolayers was confirmed by transepithelial electrical resistance measurements and by determination of the permeability of the radioactive marker polyethylene glycol (PEG4000). After 6 h approximately 30-40% of genistein and daidzein added at the apical side was transported to the basolateral side and this level was maintained for 24 h, The glycosides were barely transported through the Caco-2 cells. No significant metabolism of genistein and daidzein in the Caco-2 cells occurred, whereas the glycosides were mainly metabolised to their respective aglycones. Obviously, our data indicates that Caco-2 cells contain an endogenous glycosidase activity.

Relative bioavailability of the antioxidant flavonoid quercetin from various foods in man.

Quercetin is a strong antioxidant and a major dietary flavonoid. Epidemiological studies suggest that consumption of quercetin protects against cardiovascular disease, but its absorption in man is controversial. We fed nine subjects a single large dose of onions, which contain glucose conjugates of quercetin, apples, which contain both glucose and non-glucose quercetin glycosides, or pure quercetin-3-rutinoside, the major quercetin glycoside in tea. Plasma levels were then measured over 36 h. Bioavailability of quercetin from apples and of pure quercetin rutinoside was both 30% relative to onions. Peak levels were achieved less than 0.7 h after ingestion of onions, 2.5 h after apples and 9 h after the rutinoside. Half-lives of elimination were 28 h for onions and 23 h for apples. We conclude that conjugation with glucose enhances absorption from the small gut. Because of the long half-lives of elimination, repeated consumption of quercetin-containing foods will cause accumulation of quercetin in blood.

Structure-activity relationships between antibacterial activities and physicochemical properties of sulfonamides.

Relationships between the antimicrobial activities of sulfonamides and physicochemical properties including the acid dissociation constant (pKa) and the hydrophobicity constant (pi) were determined. The minimal inhibitory concentrations (MIC) of sulfonamides against Actinobacillus pleuropneumoniae, a gram-negative veterinary pathogen, were used. High performance liquid chromatography was applied for the determination of the electronic and hydrophobic parameters. Empirically determined relationships pointed out the dominant role of the degree of ionization on the antimicrobial activity. The data indicate that hydrophobic properties of sulfonamides, characterized by pi, are of minor importance for the in vitro antibacterial activity. Because of the restricted pKa range (4.9-7.7) it could not be established whether the relationship between pKa and activity was linear or bilinear. Whenever o,m-disubstituted sulfonamides were included correlations decreased substantially. Relationships based on multicompartment equilibrium models were derived and indicated a bilinear relation between pKa and MIC. Model-based equations showed that the antibacterial activity was governed by the extracellular ionic concentration of the sulfonamides whenever different intra and extracellular pH values were assumed in the equilibrium model. The antimicrobial activities of the sulfonamides against gram-positive organisms were also related to the degree of ionization of the sulfonamides in the agar medium.

Bioavailability of the dietary antioxidant flavonol quercetin in man.

Quercetin, a dietary antioxidant flavonoid, has anticarcinogenic properties. We quantified the absorption of quercetin in ileostomists. Absorption was 52 +/- 5% for quercetin glucosides from onions, 17 +/- 15% for quercetin rutinoside, and 24 +/- 9% for quercetin aglycone. The plasma quercetin concentration in subjects with an intact colon, after ingestion of fried onions, apples and pure quercetin rutinoside, decreased slowly with elimination half-lives of about 25 h. Thus, repeated dietary intake of quercetin will lead to accumulation in plasma. The relative bioavailability of quercetin from apples and rutinoside was one-third of that from onions. Absorption kinetics and bioavailibility might be determined by the type of glycoside. Dietary quercetin could increase the antioxidant capacity of blood plasma.

Oral absorption and metabolism of quercetin and sugar-conjugated derivatives in specific transport systems.

The intestinal transport and metabolism of quercetin and various sugar-conjugates were quantified in in vitro and in vivo model systems. The nature of the sugar moiety at the C3 and C4' position had no significant effect on the rate of transport. At the 10 microM level, quercetin and glycosides with sugars at position 3 were determined to be glucose transport carrier inhibitors.

The biotransformation of sulfadimethoxine, sulfadimidine, sulfamethoxazole, trimethoprim and aditoprim by primary cultures of pig hepatocytes.

The in vitro biotransformation of three sulfonamides, trimethoprim and aditoprim, was studied using primary cultures of pig hepatocytes. Incubation of monolayer cultures with sulfadimethoxine (SDM), sulfamethoxazole (SMX) and 14C-sulfadimidine (SDD) resulted in the formation of the corresponding N4-acetylsulfonamide to different extents, depending upon the molecular structure of the drug. Addition of the acetylsulfonamides to the cells showed that these compounds were deacetylated, each to a different extent. A relatively low degree of acetylation (in the case of SDD) was paralleled by extensive deacetylation (i.e. AcSDD), whereas extensive acetylation (i.e. SMX) was in concert with minor deacetylation (i.e. AcSMX). The addition of bovine serum albumin to the medium resulted in a decrease in conversion of sulfonamides as well as acetylsulfonamides. The main metabolic pathway of 14C-trimethoprim (TMP) was O-demethylation with subsequent conjugation. Two hydroxy (demethyl) metabolites were formed, namely 3'- and 4'-demethyl trimethoprim, which were both glucuronidated while 3'-demethyl trimethoprim was also conjugated with sulphate. The capacity to form conjugates with either glucuronic acid or sulphate was at least as high as the capacity for O-demethylation since more than 90% of the metabolites were excreted as conjugates in the urine of pigs. Addition of 14C-aditoprim (ADP) to the hepatocytes led to the N-demethylation of ADP to mono-methyl-ADP and didesmethyl-ADP. During the incubation another three unknown ADP metabolites were formed. In contrast to TMP, no hydroxy metabolites or conjugated metabolites of aditoprim were formed. These in vitro results were in agreement with the in vivo biotransformation pattern of the studied sulfonamides and trimethoprim in pigs.

Absorption and disposition kinetics of the dietary antioxidant quercetin in man.

Quercetin is a dietary antioxidant that prevents oxidation of low-density lipoproteins in vitro by scavenging to free oxygen radicals. Its intake was inversely associated with coronary heart mortality in Dutch elderly men. However, data on absorption of quercetin in man are scarce and contradictory. We studied the time course of the plasma quercetin concentration in two subjects after ingestion of fried onions containing quercetin glucosides equivalent to 64 mg of quercetin aglycone. Peak plasma levels of 196 ng/ml were reached after 2.9 h, with a half-life of absorption of 0.87 h. The half-life of the distribution phase was 3.8 h, and of the subsequent elimination phase 16.8 h. After 48 h the plasma concentration was about 10 ng/ml. We conclude that quercetin glucosides from onions are absorbed and are eliminated slowly throughout the day. Thus, the dietary antioxidant quercetin could increase the antioxidant capacity of blood plasma.

Absorption of dietary quercetin glycosides and quercetin in healthy ileostomy volunteers.

Quercetin is a dietary antioxidant that prevents oxidation of low-density lipoproteins in vitro. Intake of quercetin was inversely associated with coronary heart disease mortality in elderly Dutch men. However, the extent of absorption of quercetin in humans is unclear. The aim of this study was to quantify absorption of various forms of quercetin. Nine healthy ileostomy subjects were studied, to avoid losses caused by colonic bacteria. They followed a quercetin-free diet for 12 d; on days 4, 8, and 12 they received a supplement of fried onions at breakfast (rich in quercetin glucosides) equivalent to 89 mg aglycone, pure quercetin rutinoside (the major quercetin compound in tea) equivalent to 100 mg aglycone, or 100 mg pure quercetin aglycone, in random order. Subsequently, participants collected ileostomy effluent and urine for 13 h. In vitro incubations of quercetin or its glycosides with gastrointestinal fluids showed minimal degradation. Absorption of quercetin, defined as oral intake minus ileostomy excretion and corrected for 14% degradation within the ileostomy bag, was 52 +/- 15% for quercetin glucosides from onions, 17 +/- 15% for quercetin rutinoside, and 24 +/- 9% for quercetin aglycone. Mean excretion of quercetin or its conjugates in urine was 0.5% of the amount absorbed; quercetin excretion in urine was negatively correlated with excretion in ileostomy effluent (r = -0.78, n = 27). We conclude that humans absorb appreciable amounts of quercetin and that absorption is enhanced by conjugation with glucose.

Pharmacokinetics of sulfadimethoxine and sulfamethoxazole in combination with trimethoprim after intravenous administration to healthy and pneumonic pigs.

The pharmacokinetics of two sulfonamide/trimethoprim combinations were investigated after intravenous administration to clinically healthy pigs and to the same pigs following a challenge with Actinobacillus pleuropneumoniae toxins. Endobronchial challenge with A. pleuropneumoniae toxins resulted in fever, increased white blood cell counts and decreased water and feed consumption. Healthy, as well as febrile, pigs were given sulfadimethoxine (SDM) or sulfamethoxazole (SMX) intravenously at a dose of 25 mg/kg b.w. in combination with 5 mg trimethoprim (TMP) per kg body weight. The pharmacokinetic parameters of the sulfonamides as well as their main metabolites (acetyl sulfonamides) were not significantly different in healthy and febrile pigs. In healthy and pneumonic pigs, the mean elimination half-lives of SDM were 12.9 h and 13.4 h, respectively, those of SMX 2.5 h and 2.7 h, respectively, and those of TMP 2.8 h and 2.6 h, respectively. Distribution volumes in healthy and febrile pigs of SDM and SMX varied between 0.2 and 0.4 L/kg, and those of TMP between 1.1 and 1.6 L/kg. The mean AUC of TMP was decreased and the volume of distribution and total body clearance of TMP were increased in febrile pigs. Protein binding of the drugs and metabolites studied were not significantly changed after toxin-induced fever. The extent of protein binding of SDM, SMX and TMP was in the range 94-99%, 45-56% and 40-50%, respectively. Based on knowledge of in vitro antimicrobial activity of the drug combinations against A. pleuropneumoniae it was concluded that after intravenous administration of the dose administered (30 mg/kg of the combination preparations) to healthy and pneumonic pigs, plasma concentrations of SMX and TMP were above the concentration required for growth inhibition of 50% of A., pleuropneumoniae strains for approximately 16 h, whereas bacteriostatic plasma concentrations of SDM were still present after TMP had been eliminated from plasma. Because of similar elimination half-lives of SMX and TMP in pigs this combination is preferred to the combination of SDM with TMP.

In vitro susceptibility of some porcine respiratory tract pathogens to aditoprim, trimethoprim, sulfadimethoxine, sulfamethoxazole, and combinations of these agents.

The in vitro antimicrobial activities of aditoprim (AP), a new dihydrofolate reductase (DHFR) inhibitor, trimethoprim (TMP), sulfadimethoxine (SDM), sulfamethoxazole (SMX), and combinations of these drugs against some porcine respiratory tract pathogens were determined by use of an agar dilution method. The minimal inhibitory concentrations (MIC) of these agents were determined twice against Bordetella bronchiseptica (n = 10), Pasteurella multocida (n = 10), and Actinobacillus pleuropneumoniae (n = 20) strains isolated from pigs suffering from atrophic rhinitis or pleuropneumonia. All B bronchiseptica strains were resistant to AP and TMP. The MIC50 values of AP and TMP for P multocida were 0.25 and 0.06 microgram/ml, respectively, and for A pleuropneumoniae, 1 and 0.25 microgram/ml, respectively. The MIC50 values of SDM and SMX for B bronchiseptica were 4 and 1 micrograms/ml, respectively; for P multocida, 16 and 8 micrograms/ml, respectively; and for A pleuropneumoniae, 16 and 8 micrograms/ml, respectively. The investigated combinations of the DHFR inhibitors and the selected sulfonamides had synergism for the A pleuropneumoniae strains; the MIC90 values of the combinations were less than or equal to 0.06 microgram/ml. Potentiation was not observed for the B bronchiseptica and the P multocida isolates. The MIC of the combinations against B bronchiseptica and P multocida corresponded respectively to the concentrations of the sulfonamides and the DHFR inhibitors in the combinations. For A pleuropneumoniae, 2 types of strains were used (25% of serotype 2 and 75% of serotype 9). Type-2 strains had lower susceptibility than type-9 strains to AP and TMP as well as to SDM and SMX (at least a fourfold difference in MIC between the 2 types of strains).(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro antimicrobial activity of sulfonamides against some porcine pathogens.

The minimal inhibitory concentrations (MIC) of sulfonamides were determined against Bordetella bronchiseptica (n = 10), Pasteurella multocida (n = 10), Haemophilus pleuropneumoniae (n = 20), and Streptococcus suis (n = 10) strains isolated from pigs with atrophic rhinitis, pneumonia, or meningitis. Sulfonamides tested in an agar dilution method were sulfachloropyridazine, sulfadiazine, sulfadimethoxine, sulfamethazine, sulfadoxine, sulfisoxazole, sulfamerazine, sulfamethoxazole, sulfamethoxypyridazine, sulfanilamide, sulfatroxazole, and sulfisomidine. Results indicated that monotherapy of S suis infections with sulfonamides should not be encouraged because the MIC50 of all sulfonamides investigated was greater than 32 micrograms/ml. The MIC50 of the sulfonamides against B bronchiseptica ranged from 0.5 to 8 micrograms/ml, against P multocida from 2 to 32 micrograms/ml, and against H pleuropneumoniae from 8 to 64 micrograms/ml. The MIC50 of sulfachloropyridazine, sulfadiazine, sulfadimethoxine, sulfamerazine, and sulfamethoxazole for the gram-negative bacteria did not exceed 16 micrograms/ml. Among these compounds, sulfamethoxazole had the highest activity. The frequently prescribed sulfamethazine had an overall low antimicrobial activity.

A rapid fluorimetric screening method for chlortetracycline, oxytetracycline and tetracycline in pig meat and kidney tissues.

A rapid fluorimetric screening method for chlortetracycline, oxytetracycline and tetracycline in pig meat and kidney tissues is described. After sonication-aided extraction with ethyl acetate, the extract is cleaned and concentrated by means of solid-phase extraction using an aromatic sulphonic acid cation-exchange column. Subsequent screening is carried out by fluorimetry. The detection level is estimated to be about 0.05 mg kg-1 for oxytetracycline, 0.1 mg kg-1 for chlortetracycline and 0.2 mg kg-1 for tetracycline.

Determination of sulfadimethoxine, sulfamethoxazole, trimethoprim and their main metabolites in porcine plasma by column switching HPLC.

A HPLC method for the determination of sulfadimethoxine, sulfamethoxazole, trimethoprim and their main metabolites in porcine plasma is reported. The metabolites under investigation were the N4-acetyl sulfonamides and 3'- and 4'-demethyl trimethoprim. In order to obtain a sensitivity of 25-50 ng ml-1, the application of column switching HPLC was investigated. An on-line preconcentration of the drugs and metabolites was preceded by an off-line sample pre-treatment. Parent compounds and metabolites were separated by reversed-phase HPLC followed by UV-detection. The mean recoveries for 4'-demethyl trimethoprim were greater than 80% while the mean recoveries for the other compounds were greater than 90%. Application of the method for analysis of plasma samples obtained from pharmacokinetic studies is described.