Risk management-an industry approach.

An effective risk management system covering the whole process of food production from "farm to fork" is required by the food industry in order to assure that the food provided to consumers is safe. Food safety and quality assurance begins with the design and development of food products starting with product conceptualisation and continuing with the selection, purchasing, and evaluation of raw materials and with the specifications for processing, packaging and distribution. Within a larger quality management framework a number of tools have been developed by the food industry, which when used in an integrated fashion facilitate the management of food safety. These include good manufacturing practice (GMP), good hygiene practice (GHP) and HACCP (hazard analysis critical control point) as well as quality systems which allow the verification that all factors affecting the safety of a product are under control. Finally, regulations and systems can only function if they are applied. Everyone, from the farmer, the line operator in the manufacturing plant, to the person handling the food in distribution and sales, needs to be aware of his influence with regards safety. The effectiveness of safety awareness programs specific to each area is key to an industry approach to risk management.

The coffee-specific diterpenes cafestol and kahweol protect against aflatoxin B1-induced genotoxicity through a dual mechanism.

The diterpenes cafestol and kahweol (C&K) have been identified in animal models as two potentially chemoprotective agents present in green and roasted coffee beans. It has been postulated that these compounds may act as blocking agents by producing a co-ordinated modulation of multiple enzymes involved in carcinogen detoxification. In this study, we investigated the effects of C&K against the covalent binding of aflatoxin B1 (AFB1) metabolites to DNA. Male Sprague-Dawley rats were treated with increasing amounts of a mixture of C&K in the diet (0-6200 p.p.m.) for 28 and 90 days. A dose-dependent inhibition of AFB1 DNA-binding was observed using S9 and microsomal subcellular fractions from C&K-treated rat liver in an in vitro binding assay. Significant inhibition was detected at 2300 p.p.m. and maximal reduction of DNA adduct formation to nearly 50% of the control value was achieved with 6200 p.p.m. of dietary C&K. Two complementary mechanisms may account for the chemopreventive action of cafestol and kahweol against aflatoxin B1 in rats. A decrease in the expression of the rat activating cytochrome P450s (CYP2C11 and CYP3A2) was observed, as well as a strong induction of the expression of the glutathione-S-transferase (GST) subunit GST Yc2, which is known to detoxify highly the most genotoxic metabolite of AFB1. These data and the previously demonstrated effects of C&K against the development of 7,12-dimethylbenz[a]anthracene (DMBA)-induced carcinogenesis at various tissue sites suggest the potential widespread effect of these coffee components against chemical carcinogenesis.

The ADI as a basis to establish standards for pesticide residues in food products for infants and children.

Pesticides are widely used in agriculture to control a variety of detrimental organisms. As a consequence, low but measurable amounts of residues may be found in the food supply including food intended for infants and young children. This has been the cause of some alarm since it is difficult for the general public to understand the magnitude of health risk associated with the consumption of food contaminated with low levels of potentially toxic chemicals. In this context safety-based regulations for pesticide residues that ensure the protection of infants and young children are of crucial importance. In this article we discuss the applicability of the ADI to infants and children with regards to pesticides and outline a proposal which has been devised to establish residue limits for finished baby food products.

Effect of a coffee lipid (cafestol) on cholesterol metabolism in human skin fibroblasts.

Consumption of boiled coffee promotes an elevation of plasma cholesterol concentration in humans. The active compounds found in the lipid fraction of the coffee have been identified as the diterpenes cafestol and kahweol. We have studied the effects of pure cafestol on cholesterol metabolism in human skin fibroblasts (HSF). The uptake of [125I]-labeled tyramine cellobiose-labeled low density lipoprotein ([125I]TC-LDL) was decreased by about 50% (P< 0.05) after 18 h preincubation time with cafestol (20 microg/ml), as compared to the control cells. The specific binding of radiolabeled LDL was reduced by 54% (P < 0.05) after preincubation for 18 h with cafestol. A reduced amount of LDL receptors was demonstrated by a protein-normalized Scatchard plot analysis (20% decrease in Bmax) as well as by immunoblotting (25%) after cafestol incubation. No significant effect was observed on the level of mRNA for the LDL receptor after 11 and 23 h incubation with cafestol. Furthermore, we transfected HSF cells with a promoter region for the LDL receptor gene linked to a reporter gene, chloramphenicol acetyl transferase (CAT). No change was seen in the CAT activity after incubation with cafestol (20 microg/ml). Moreover, cafestol caused a 2.3-fold (P < 0.05) higher incorporation of radiolabeled [14C]oleic acid into cholesteryl esters after 24 h incubation, as compared to control cells, suggesting an increased acyl-CoA:cholesterol acyl transferase (ACAT) activity. Incorporation of [14C]acetate into cholesterol was reduced by approximately 40% (P < 0.05) with cafestol (20 microg/ml), as compared to control after 24 h preincubation, indicating a decreased 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase activity. Our results suggest that intake of cafestol may cause increased concentration of plasma cholesterol via the down-regulation of low density lipoprotein receptors by post-transcriptional mechanisms.

Effect of coffee lipids (cafestol and kahweol) on regulation of cholesterol metabolism in HepG2 cells.

We studied the effect of the coffee diterpene alcohols, cafestol and kahweol, on cholesterol metabolism in HepG2 cells. Uptake of 125I-tyramine cellobiose-labeled LDL was decreased by 15% to 20% (P < .05) after 18 hours of preincubation with cafestol (20 micrograms/mL), whereas 25-hydroxycholesterol reduced uptake by 55% to 65% (P < .05). Degradation of LDL in the presence of cafestol was decreased by 20% to 30% (P < .05) under the same conditions. The effect of cafestol (20 micrograms/mL) on uptake and degradation of LDL was greatest (35% to 40%, P < .05) after 6 and 10 hours of preincubation, respectively. Furthermore, the effect of cafestol was also dependent on its concentration, and a significant decrease in the LDL uptake (19%) was observed at 10 micrograms/mL (P < .05). Specific binding of LDL was reduced by 17% (P < .05) and 60% (P < .05) after preincubation with cafestol (20 micrograms/mL) and 25-hydroxycholesterol (5 micrograms/mL) for 6 hours, respectively, compared with control cells. Analysis of LDL binding showed that cafestol reduced the number of binding sites for LDL on the cell surface (capacity) by 35% (P < .05). In contrast, no significant effect on the level of mRNA for the LDL receptor was observed after incubation with cafestol, whereas 25-hydroxycholesterol reduced the mRNA level for the LDL receptor by 40% to 50% (P < .05). A fusion gene construct consisting of a synthetic sterol regulatory element-1 (SRE-1) promoter for the human LDL receptor coupled to the reporter gene for chloramphenicol acetyltransferase (CAT) was transfected into HepG2 cells. No change was observed in CAT activity in SRE-1-transfected cells after incubation with cafestol, whereas 25-hydroxycholesterol reduced CAT activity by 30% to 40% (P < .05). Incorporation of [14C]acetate into unesterified cholesterol and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity were unaffected in cells incubated with cafestol as well as the cafestol-kahweol mixture compared with control cells. Moreover, cafestol and the cafestol-kahweol mixture did not promote increased incorporation of radiolabeled [14C]oleic acid into cholesteryl esters after short-term incubation compared with control cells. On the other hand, 25-hydroxycholesterol caused a 70% to 90% reduction of cholesterol synthesis (P < .05) and HMG-CoA reductase activity (P < .05), decreased HMG-CoA reductase mRNA level by 70% to 80% (P < .05), and promoted a twofold increase in cholesterol esterification (P < .05). Finally, no effect of the coffee diterpenes on bile acid formation was observed. These results suggest that cafestol (and kahweol) may reduce the activity of hepatic LDL receptors and thereby cause extracellular accumulation of LDL.

Analysis of the content of the diterpenes cafestol and kahweol in coffee brews.

The diterpenes cafestol and kahweol have been implicated as the components in boiled coffee responsible for its hypercholesterolaemic effects. These particular coffee constituents have also been shown to possess anticarcinogenic effects. A simple and sensitive reverse-phase HPLC method using solid-phase extraction has been developed for the analysis of cafestol and kahweol in coffee brews. This method was used to confirm that the method of coffee brewing is a major determinant of the cup content and hence level of consumption of these diterpenes. Scandinavian-style boiled coffee and Turkish-style coffee contained the highest amounts, equivalent to 7.2 and 5.3 mg cafestol per cup and 7.2 and 5.4 mg kahweol per cup, respectively. In contrast, instant and drip-filtered coffee brews contained negligible amounts of these diterpenes, and espresso coffee contained intermediate amounts, about 1 mg cafestol and 1 mg kahweol per cup. These findings provide an explanation for the hypercholesterolaemic effect previously observed for boiled coffee and Turkish-style coffee, and the lack of effect of instant or drip-filtered coffee brews. This methodology will be of value in more correctly assessing the human exposure to these diterpenes through the consumption of coffee, and hence the potential physiological effects of different brews.

Effects of the naturally occurring food mycotoxin ochratoxin A on brain cells in culture.

The potential of ochratoxin A (OTA) to damage brain cells was studied by using a three-dimensional cell culture system as model for the developing brain. Aggregating cell cultures of foetal rat telencephalon were tested either during an early developmental period, or during a phase of advanced maturation, over a wide range of OTA concentrations (0.4 nM to 50 microM). By monitoring changes in activities of cell type-specific enzymes (ChAt and GAD, for cholinergic and GABAergic neurones, respectively, GS for astrocytes and CNP for oligodendrocytes), the concentration-dependent toxicity and neurodevelopmental effects of OTA were determined. OTA proved to be highly toxic, since a 10-day treatment at 50 nM caused a general cytotoxicity in both mature and immature cultures. At 10 nM of OTA, cell type-specific effects were observed: in immature cultures, a loss in neuronal and oligodendroglial enzyme activities, and an increase in the activity of the astroglial marker glutamine synthetase were found, Furthermore, at 2 and 10 nM of OTA, a clustering of microglial cells was observed. In mature cultures, OTA was somewhat less potent, but caused a similar pattern of toxic effects. A 24 h-treatment with OTA resulted in a concentration-dependent decrease in protein synthesis, with IC50 values of 25 nM and 33 nM for immature and mature cultures respectively. Acute (24 h) treatment at high OTA concentrations (10 to 50 microM) caused a significant increase in reactive oxygen species formation, as measured by the intracellular oxidation of 2',7'-dichlorofluorescin. These results suggest that OTA has the potential to be a potent toxicant to brain cells, and that its effects at nanomolar concentrations are primarily due to the inhibition of protein synthesis, whereas ROS seem not to be involved in the toxicity mediated by a chronic exposure to OTA at such low concentrations.

Placental glutathione S-transferase (GST-P) induction as a potential mechanism for the anti-carcinogenic effect of the coffee-specific components cafestol and kahweol.

The coffee specific diterpenes cafestol and kahweol (C + K) have been reported to be anti-carcinogenic in several animal models. It has been postulated that this activity may be related to their ability to induce glutathione S-transferases (GSTs). We investigated the influence of a mixture of C + K, incorporated at various levels in the diet of Sprague-Dawley rats, on the expression of different hepatic GST iso-enzymes. Liver samples were examined using isoform-specific GST substrates and antibodies, and highly selective oligomers were employed to determine effects at the RNA level. A dose-dependent increase in general GST activity was observed in male and female animals following 28 or 90 days of treatment. A time-course study demonstrated that the maximal effect was observed within 5 days of treatment. Little or no effect was found on the activity of GST alpha and mu iso-enzymes. The most striking observation was a dose-dependent induction of placental glutathione S-transferase (GST-P) which could be demonstrated at the mRNA, protein and enzymatic levels. This effect was observed in both male and female rats. The maximal induction was attained within 5 days of treatment with C + K, remained elevated with continued treatment, but was reversible on withdrawal of treatment. Immunohistochemical examination of liver slices revealed a strong even distribution of GST-P expression throughout the acinus at the highest dose of C + K, while at lower doses the induction of GST-P occurred predominantly in periportal hepatocytes. There was no indication of the presence of preneoplastic foci and, furthermore, the effect of C + K on the GST-P was completely reversible. These findings indicate that the anticarcinogenic mechanism of C + K may involve a specific induction of GST-P and suggest a potential role for GST-P in detoxifying carcinogenic compounds.

Limits for pesticide residues in infant foods: a safety-based proposal.

A review of safety procedures for regulating pesticide residues in food commodities has demonstrated that maximum residue levels established by national and international bodies may not be suitable for direct application to finished infant products. We have developed a safety-based strategy specifically aimed at setting limits for pesticide residues in baby foods. The approach is based on the acceptable daily intake (ADI) together with the application, when necessary, of an additional uncertainty factor to account for the potential higher sensitivity of infants to toxicants. The need for this extra factor is dependent on the toxicological information available and may be particularly important for pesticides with a neurotoxic potential. Using this strategy we have evaluated safety-based residue limits for finished products based on a standard food intake pattern of a 4-month-old infant. For most of the pesticides examined the estimated limits fell within a range which can be controlled through existing quality assurance systems. This scientific approach appears usable in practice and is intended as a basis for stimulating discussions aimed at evaluating the need for new limits to ensure the optimal quality of infant products with respect to pesticide safety. This assessment demonstrates that for a number of pesticides there is a need for adequate developmental studies to provide assurance that the current ADI is appropriate for infants. A consequence may be the necessity for enhanced analytical sensitivity for the determination of pesticide residues in infant foods which would be compatible with limits based on an infant-adjusted ADI.

Effect of a coffee lipid (cafestol) on regulation of lipid metabolism in CaCo-2 cells.

The influence of cafestol, a lipid component found in boiled coffee, on low density lipoprotein (LDL) and lipid metabolism was investigated in CaCo-2 cells cultured on filter membranes. The rate of uptake and degradation of 125I-labeled tyramine cellobiose-LDL was increased 50% in CaCo-2 cells incubated with cafestol (20 micrograms/ml, 63 microM) for 24 h, whereas in cells incubated with 25-hydroxycholesterol (10 micrograms/ml, 25 microM) the rate of uptake and degradation showed a 30% decrease. A mixture of kahweol and cafestol, both natural components of coffee beans, modestly enhanced the rate of LDL uptake and degradation, as compared to pure cafestol. Incubation of cafestol with CaCo-2 cells induced a 3-fold up-regulation of LDL receptor mRNA, as compared to control cells. In contrast, incubation of the cells with 25-hydroxycholesterol produced a 30% decrease of LDL receptor expression. CaCo-2 cells were transfected with a promoter region containing the sterol regulatory element-1 (SRE-1) coupled to the reporter gene chloramphenicol acetyltransferase (CAT). When cells transfected with SRE-1 promoter were incubated with cafestol, there was a 20% up-regulation of CAT activity, whereas 25-hydroxycholesterol abolished this activity. Cafestol contributed to a significantly lowered secretion of cholesteryl ester and triacylglycerol, regardless of the radiolabeled precursor used ([2-14C]acetic acid, [1,2,3-3H]glycerol, [3H]water, and [1-14C]oleic acid). This reduction in secretion of lipids was accompanied by an increase in trichloroacetic acid-soluble activity when radiolabeled oleic acid was used as a tracer. We conclude that cafestol promotes an enhanced rate of uptake and degradation of LDL, probably due to an increase in transcription of LDL receptor mRNA and a reduced secretion of cholesteryl ester and triacylglycerol in CaCo-2 cells.

Expression of MHC antigens by intestinal epithelial cells. Effect of transforming growth factor-beta 2 (TGF-beta 2).

The effect of interferon-gamma (IFN-gamma) and TGF-beta 2 on expression of MHC antigens by the human intestinal epithelial cell line HT-29 was examined by immunohistochemistry and flow cytometry. Untreated HT-29 cells constitutively expressed HLA-ABC but little HLA-DR. Expression of both molecules was increased by IFN-gamma (100 U/ml, 24 h). TGF-beta 2 at concentrations > or = 0.5 ng/ml given before or simultaneously with IFN-gamma, inhibited the IFN-induced expression of HLA-DR. Small increases in HLA-ABC expression by IFN-gamma were further increased by pretreatment with TGF-beta 2, while a strong induction of HLA-ABC was inhibited by the TGF-beta 2 pretreatment. Our results suggest that the inhibitory action of the TGF-beta 2 on both HLA-ABC and HLA-DR correlates with the degree of induction following IFN-gamma treatment. Since TGF-beta 2 is present in milk and is produced by gut epithelial cells, one of its possible functions may be to regulate expression of HLA antigens in the neonatal intestine and/or diseased intestine.

Polymeric nutrition as the primary therapy in children with small bowel Crohn's disease.

BACKGROUND: Recent studies in adults have shown that polymeric (whole protein) diets are as effective as semi-elemental and elemental formulae for the induction of remission in small bowel Crohn's disease. Whole protein diets are more palatable and cheaper. There have been no studies confirming efficacy in children. PATIENTS AND METHODS: We report our experience with seven children with active small bowel Crohn's disease given a casein-based, polymeric feed rich in TGF-beta 2 (Specific Polymeric Diet; Nestle-Clintec; Vevey, Switzerland) as complete nutrition for 8 weeks. RESULTS: Initial and follow-up assessments were performed. All children showed a significant improvement in disease activity, with C-reactive protein returning to normal, an increase in serum albumin and a good weight gain. Initial and follow-up ileal biopsies were assessed and showed reduced mucosal inflammation in six of seven children, with complete healing in two. CONCLUSION: In an uncontrolled descriptive study we have shown that a polymeric (whole protein) diet is a therapeutic option for small bowel Crohn's disease in children. By comprehensive follow-up we have demonstrated clinical and biochemical remission, with an improved endoscopic appearance and a reduction of mucosal inflammation in the terminal ileum.

Identity of the cholesterol-raising factor from boiled coffee and its effects on liver function enzymes.

Boiled coffee contains an unidentified lipid that raises serum cholesterol. We studied the effects of the ingestion of coffee oil fractions of increasing purity in volunteers in order to identify the cholesterol-raising factor. In 15 volunteers who ingested 0.75 g/d of a non-triglyceride-fraction from coffee oil for 4 weeks, mean cholesterol increased by 48 mg/dl (1.2 mmol/l) relative to placebo. In contrast, a coffee oil stripped of the non-triglyceride lipids cafestol and kahweol had no effect. In three volunteers, purified cafestol (73 mg/d) plus kahweol (58 mg/d) increased cholesterol by 66 mg/dl (1.7 mmol/l) after 6 weeks. Oil from Robusta beans, which contains cafestol but negligible kahweol, also raised serum cholesterol. These findings show that cafestol is at least partly responsible for the cholesterol-raising effect of boiled coffee. Coffee oils and brews containing cafestol consistently increased serum triglycerides and alanine amino-transferase, and depressed serum creatinine and gamma-glutamyl-transferase (GGT). After withdrawal GGT activity rose above baseline. Norwegians who habitually consumed 5-9 cups of boiled coffee per day had higher serum cholesterol levels and lower GGT but no higher alanine aminotransferase activity than controls. Thus, serum cholesterol is raised by cafestol and possibly also kahweol, both natural components of coffee beans. The mechanism of action is unknown but is accompanied by alterations in liver function enzymes.

Pathology of spontaneous and oncogene transformed rat liver epithelial cells and derived tumours in nude mice.

The histological and ultrastructural features of oncogene transformed rat liver epithelial (RLE) cells in culture and spontaneously transformed RLE cells were studied. The tumours produced in nude mice by all the transformed cells were either moderately well differentiated carcinomas or sarcomatous tumours. Epithelial tumours were produced in the RLE cells after transduction of both v-raf and v-myc oncogenes whereas sarcomatous tumours were produced after transduction of v-raf alone. These data suggested that transformation of RLE cells with a single oncogene, v-raf, produced malignant tumours which were consistent with sarcomas while a combination of two oncogenes, v-raf and v-myc, produced an epithelial tumour, consistent with a carcinoma. The effects of these oncogenes on RLE cells indicate that they were able to differentiate and were capable of producing two morphologically distinct tumour types. The possible role of v-myc in switching the sarcomatous lineage to an epithelial tumour lineage is considered to be significant and worthy of further studies. The epithelial tumour produced in RLE cells by combination of v-raf and v-myc is consistent with an embryonal type of hepatoblastoma. The trabecular type of liver cell carcinoma which resulted from spontaneous transformation of RLE cells illustrates the inherent potential of the RLE cell to undergo malignant change and strongly suggests that the RLE cells may be the precursor cells in the development of hepatocellular carcinoma in the rat.

Homologous and heterologous gap-junctional intercellular communication in v-raf-, v-myc-, and v-raf/v-myc-transduced rat liver epithelial cell lines.

We examined gap-junctional intercellular communication (GJIC) in a series of normal and v-raf-, v-myc-, and v-raf/v-myc-transduced rat liver epithelial (RLE) cell lines using the scrape loading-dye transfer and fluorescence-recovery-after-photobleaching (FRAP) assays. Whereas the normal RLE cell line, the control helper virus-transduced cell line, and the v-myc-transduced cell line all showed excellent GJIC, the v-raf-transduced cell lines displayed decreasing levels of GJIC associated with their increasing tumorigenicity. The v-raf/v-myc-transformed cell lines showed the lowest levels of GJIC and were also the most tumorigenic. Heterologous GJIC of these oncogene-transduced cell lines was also compared with that in the normal RLE cells. A modified FRAP assay, using fluorescent-microbead labelling to identify the oncogene-transduced cell from surrounding normal cells, was used to quantify the heterologous GJIC. The v-raf/v-myc-transformed RLE cells had no heterologous communication with the normal RLE cells, whereas v-raf- and v-myc-transduced cell lines maintained heterologous GJIC. Northern analysis showed that connexin 43 was the only gap-junction protein message expressed in these cell lines; connexin 32 and connexin 26 were not expressed. The levels of connexin 43 mRNA expression were relatively unchanged in all cell lines, suggesting that the reduction in GJIC was primarily at the posttranslational level. These findings suggest that reduction of homologous GJIC in v-raf- and v-raf/v-myc-transformed RLE cells is linked to their tumorigenic potential. Furthermore, the loss of heterologous GJIC, which we observed only in the v-raf/v-myc-transformed cells, might release such cells from the growth-regulating effects of surrounding normal cells, possibly contributing to their enhanced tumorigenic potential.