Assessment of occupational and dietary exposure to pesticide residues.

Plant protection products (PPPs) are pesticides containing at least one active substance that drives specific actions against pests (diseases). PPPs are regulated in the EU and cannot be placed on the market or used without prior authorisation. EFSA assesses the possible risks of the use of active substances to humans and environment. Member States decide whether or not to approve their use at EU level. Furthermore, Member States decide at national level on the authorisation of PPPs containing approved substances. In agriculture, exposure to PPPs and their residues during occupational tasks is estimated prior to product authorisation, using models fed with study-specific (e.g. absorption, dissipation) and default values. Exposure of workers to pesticide residues reduces with the pesticide's dissipation time during crop-related tasks. However, the current risk assessment gap is that no methodology is available to calculate the re-entry interval (REI) for workers, which specifies how long they should wear personal protective clothing during their first entry into pesticide-sprayed crops. Protective clothing (such as gloves) can reduce pesticide residue exposure to an acceptable level of worker safety. Within the European Food Risk Assessment Fellowship Programme (EU-FORA) assignment, a methodology was developed to calculate agricultural-use-specific and pesticide-specific REIs for which period workers should wear gloves. This was an assignment of the Dutch Ministry of Social Affairs and Employment. Another important aspect of risk assessment to ensure consumer safety is dietary risk assessment. A critical evaluation of residue studies and metabolism of the pesticide in question in crops results in a residue definition for dietary risk assessment and for enforcement and monitoring to define maximum residue limits allowed legally on or in raw agricultural commodities when applying pesticides according to good agricultural practices. This work was assigned by the Dutch Ministry of Health, Welfare and Sport and contributes to the work of the Joint FAO/WHO Meeting on Pesticide Residues.

Sterol carrier protein 2 (non-specific lipid transfer protein) is localized in membranous fractions of Leydig cells and Sertoli cells but not in germ cells.

The cellular and subcellular distribution of sterol carrier protein 2 (SCP2; nsL-TP) was reinvestigated in rat testicular cells by Western blotting and immunocytochemistry, using the affinity purified antibody against rat liver SCP2. Western blot analysis revealed high levels of the protein in the somatic cells of the testis, e.g., Leydig and Sertoli cells whereas it could not be detected in germ cells. This cellular localization of SCP2 was confirmed by Northern blotting. Immunocytochemical techniques revealed that in Leydig cells, immunoreactive proteins were concentrated in peroxisomes. Although SCP2 was also detected in Sertoli cells, a specific subcellular localization could not be shown. SCP2 was absent from germ cells. Analysis of subcellular fractions of Leydig cells showed that SCP2 is membrane bound without detectable amounts in the cytosolic fraction. These results are at variance with data published previously which suggested that in Leydig cells a substantial amount of SCP2 was present in the cytosol and that the distribution between membranes and cytosol was regulated by luteinizing hormone. The present data raise the question in what way SCP2 is involved in cholesterol transport between membranes in steroidogenic cells but also in non-steroidogenic cells.

The precursor form of the rat liver non-specific lipid-transfer protein, expressed in Escherichia coli, has lipid transfer activity.

The cDNA encoding the precursor form of non-specific lipid-transfer protein (pre-nsL-TP) from rat liver was cloned into the expression vector pET3d. The resulting plasmid was transformed to the Escherichia coli strain BL21(DE3). After induction of the bacteria with isopropyl-beta-D-thiogalactopyranoside (IPTG) pre-nsL-TP was purified from the bacterial lysate by anion exchange chromatography followed by gelfiltration. From 11 of culture, 6-7 mg of pre-nsL-TP was obtained, equal to approximately 7% of the cytoplasmic protein. By use of a fluorescence lipid transfer assay, pre-nsL-TP was found to have lipid transfer activity identical to mature nsL-TP.

Subcellular localisation and processing of non-specific lipid transfer protein are not aberrant in Rhizomelic Chondrodysplasia Punctata fibroblasts.

The import into peroxisomes and maturation of peroxisomal 3-oxoacyl-CoA thiolase are impaired in patients with the Rhizomelic form of Chondrodysplasia Punctata (RCDP). Here we show by means of immunoblotting and subcellular fractionation that non-specific lipid transfer protein (nsLTP), another peroxisomal protein synthesised as a larger precursor, is localised in peroxisomes and is present as the mature protein in RCDP fibroblasts. Thus the component of the import machinery defective in RCDP is not required for the import of nsLTP into peroxisomes.

The non-specific lipid-transfer protein (sterol carrier protein 2) and its relationship to peroxisomes.

The non-specific lipid-transfer protein (nsL-TP), also known as sterol carrier protein 2 (SCP2), is a small (M(r) 13,000) basic protein which catalyzes in vitro the transfer of a great variety of lipids, including cholesterol, between membranes. Inherent to this transfer activity, the protein stimulates in vitro various aspects of cholesterol metabolism. nsL-TP is synthesized as a precursor (pre-nsL-TP) with a leader sequence of 20 amino acid residues. It appears that the peroxisomes play an important role in the conversion of pre-nsL-TP into the mature form. In fact, nsL-TP appears to be mainly present in peroxisomes as shown by immunogold labeling of rat liver, adrenals and testes using the anti-nsL-TP antibody. However, interpretation of the data is complicated by the fact that the antibody raised against nsL-TP also reacts with a protein with a M(r) of 58,000. From cDNA analysis it became apparent that the cross-reactive 58-kDa protein contains the complete sequence of pre-nsL-TP at its C-terminus. However, pre-nsL-TP and the 58-kDa protein are synthesized from different mRNAs. Interestingly, the N-terminal part of the 58-kDa protein was found to have significant sequence similarity with 3-oxoacyl-CoA thiolase. Both pre-nsL-TP and the 58-kDa protein contain the C-terminal peroxisomal targeting tripeptide Ala-Lys-Leu. However, as shown by subcellular fractionation studies the 58-kDa protein is exclusively localized in the peroxisomes whilst nsL-TP is not only detected in the peroxisomes but also in other subcellular fractions. Moreover, a membrane-bound form of nsL-TP was detected. This membrane-bound form is present at the cytosolic side of the membranes. The physiological function of nsL-TP is still unclear; some recent developments are discussed briefly in the last part of this review.

The amino acid sequence of rat liver non-specific lipid transfer protein (sterol carrier protein 2) is present in a high molecular weight protein: evidence from cDNA analysis.

The affinity-purified antibody against rat liver non-specific lipid transfer protein (nsL-TP; sterol carrier protein 2) was used to screen a lambda-gt11 rat liver cDNA library. Positive cDNA clones were further identified by Southern blot analysis and sequenced. The largest cDNA clone consisted of 1851 bp starting at the 5' end with an open reading frame of 1545 bp. The 369 bp located at the 3' end of this open reading frame corresponded with the amino acid sequence of nsL-TP.

Identification of the non-specific lipid-transfer protein (sterol carrier protein 2) in peroxisomes of lung type II cells.

In view of the proposed role of the non-specific lipid-transfer protein (nsL-TP; sterol carrier protein 2) in the metabolism of pulmonary surfactant lipids (Batenburg et al. (1994) Biochem. J. 298, 223-229), its subcellular localization was studied in the surfactant producing alveolar type II cells. It was shown by immuno-electron microscopy that nsL-TP colocalizes with the peroxisomal marker catalase. The peroxisomal localization of nsL-TP was confirmed by gradient fractionation of type II cell homogenates. As a peroxisomal marker acyl-CoA:dihydroxyacetone-phosphate acyltransferase was assayed. Given this subcellular localization, it is very unlikely that nsL-TP plays a role in the transfer of surfactant lipids from the endoplasmic reticulum to the lamellar bodies. These results strengthen the opinion that peroxisomes are involved in surfactant synthesis.

Tissue-specific distribution of a peroxisomal 46-kDa protein related to the 58-kDa protein (sterol carrier protein x; sterol carrier protein 2/3-oxoacyl-CoA thiolase).

The complete sequence of the nonspecific lipid-transfer protein (nsL-TP; sterol carrier protein 2) including the presequence is present at the C-terminus (residues 405-547) of a 58-kDa protein. To be able to study this 58-kDa protein without the interference of nsL-TP, antibodies were raised against predicted epitope regions in the N-terminal part (peptide I, residues 23-43; peptide II, residues 130-149). Using these antibodies, rat tissues were analyzed by immunoblotting. In rat liver, in addition to the 58-kDa protein the antibody against peptide I (alpha-58K23) as well as the antibody against peptide II (alpha-58K130) detected a 46-kDa protein. This suggests that both peptide sequences are present in this 46-kDa protein. Both the 46- and the 58-kDa-proteins were abundantly present in liver and adrenals, but could also be detected in brain, kidney, heart, lung, testes, and ovary. This distribution was observed in tissues from both male and female rats. Immunogold labeling of cryosections of liver showed that alpha-58K23 labels the peroxisomes. From double-labeling experiments using alpha-nsL-TP and alpha-58K23 we conclude that the 46-kDa protein is peroxisomal. We propose that in the peroxisomes the protease that processes pre-nsL-TP also cleaves the 58-kDa protein giving rise to the 46-kDa protein and nsL-TP. In addition to the 58- and 46-kDa proteins, an immunoreactive 44-kDa protein was prominently present in rat heart and at low levels also in small intestine and brain. Immunogold labeling of cryosections of heart and Western blotting of purified mitochondria showed that the 44-kDa protein is localized in the mitochondria. The 44-kDa protein was shown to be identical to mitochondrial sarcomeric creatine kinase, which has a peptide segment of five amino acid residues in common with peptide I.

Phospholipid-transfer proteins and their mRNAs in developing rat lung and in alveolar type-II cells.

Gene expression of non-specific lipid-transfer protein (nsL-TP; identical with sterol carrier protein 2) and phosphatidylinositol-transfer protein (PI-TP) was investigated in developing rat lung. During the late prenatal period (between days 17 and 22) there is a 7-fold increase in the level of nsL-TP and a 2-fold rise in that of PI-TP. The prenatal increases in the levels of nsL-TP and PI-TP are accompanied by parallel increases in the levels of their mRNAs, indicating pretranslational regulation. Compared with whole lung, isolated alveolar type-II cells are enriched in nsL-TP and its mRNA, but not in PI-TP and its mRNA. The observation that the levels of nsL-TP and its mRNA in rat lung show a pronounced increase in the period of accelerated surfactant formation, together with the observation that the surfactant-producing type-II cells are enriched in nsL-TP and its mRNA, suggest that nsL-TP plays a role in the metabolism of pulmonary surfactant.

Identification of the cDNA clone which encodes the 58-kDa protein containing the amino acid sequence of rat liver non-specific lipid-transfer protein (sterol-carrier protein 2). Homology with rat peroxisomal and mitochondrial 3-oxoacyl-CoA thiolases.

The relationship between the rat liver non-specific lipid-transfer protein (nsLTP) and the 58-kDa protein cross-reactive with anti-nsLTP antibodies, was investigated by cDNA analysis. A 1945-bp cDNA clone was isolated which encodes a 58.7-kDa protein. This protein is identical to the 58-kDa immunoreactive protein determined by N-terminal sequence analysis of the purified 58-kDa protein. It consists of 546 amino acid residues, of which the 123 C-terminal residues are identical to the sequence of nsLTP. The N-terminal 400 amino acid residues of the 58.7-kDa protein were found to have 23.5% identity with the sequence of both mitochondrial and peroxisomal rat 3-oxoacyl-CoA thiolases, including a hypothetical substrate-binding site. The cDNA insert hybridizes with 1.1-kb, 1.7-kb, 2.4-kb and 3.0-kb mRNA species in RNA isolated from various rat tissues and from Chinese hamster ovary (CHO) cells. Southern blot analysis suggests that these mRNA species are generated from a single gene. Mutant CHO cells, deficient in peroxisomes, lack nsLTP. We have found that the mRNA encoding nsLTP is still present in these cells, which suggests that the absence of this protein is related to the lack of peroxisomes.