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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.

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Two genes, MDR1 and MDR3, constitute the human P-glycoprotein gene family. To examine the evolutionary relationship between the three known classes of mammalian P-glycoprotein genes, we have cloned the MDR3 gene and compared its structure with that of the human MDR1 and the mouse mdr1 (mdr1b) genes analyzed by other groups. The MDR3 gene contains 28 exons and 27 of these contain coding sequences for the two homologous halves of the protein that correlate with functional domains. This structure is virtually identical to that of the human MDR1 gene and the mouse mdr1 (mdr1b) gene, indicating that the exon/intron structure was fixed before the duplication events that generated different classes of P-glycoproteins, but after the P-glycoproteins diverged from related genes, like the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which has an entirely different exon/intron structure. The four alternatively spliced transcripts of the MDR3 gene arise from alternative splicing of exons 23 and 26. Our analysis of DNA clones covering about 120 kilobases (kb) of the human MDR locus, including the entire MDR3 gene (74 kb) and the intergenic region between both genes (34 kb), combined with pulsed-field gel electrophoresis data shows that the human MDR locus covers about 230 kb. In contrast to the mouse mdr genes, both human genes are transcribed in the same direction (MDR3 located downstream of MDR1). The CpG-rich sequences marking the 5' ends of both genes are hypomethylated to different extents in different cell lines. Hypomethylation roughly correlates with transcriptional activity.

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We have transfected a eukaryotic expression vector containing a mdr1 complementary DNA isolated from normal human liver into human BRO melanoma cells to study the drug-resistant phenotype produced by the exclusive overexpression of normal human mdr1 P-glycoprotein. The drug resistance pattern of mdr1-transfected clones includes relatively high resistance to gramicidin D (about 300-fold), vincristine (about 100-fold), and actinomycin D (about 100-fold) and a lower degree of resistance to doxorubicin (about 10-fold), VP16-213 (about 10-fold), and colchicine (about 6-fold). The transfectants did not exhibit resistance to trimetrexate, cis-platinum, mitomycin C, 1-beta-D-arabinofuranosylcytosine, bleomycin, G418, or magainin-2-amide; they were slightly more sensitive to verapamil (2-fold) but not to Triton X-100. As in other multidrug-resistant cell lines, resistance to vincristine could be reversed by verapamil and, more effectively, by cyclosporin A. Chloroquine only marginally increased drug sensitivity in mdr1-transfected cells. Gramicidin D resistance was also reversed by verapamil, suggesting that the mechanism of resistance to this polypeptide antibiotic is similar to that of other drugs transported by P-glycoprotein. Thus, expression of the wild-type mdr1 complementary DNA induces a drug-resistant phenotype similar to that induced by mdr1 complementary DNAs isolated from drug-resistant cell lines with relatively low colchicine resistance. As other cell lines may display a different pattern of drug resistance, it is clear that other resistance mechanisms or cell type-specific factors may modulate the resistance. mdr1-transfected cell lines provide a convenient tool for the identification of P-glycoprotein-mediated phenomena.

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Multidrug resistance (MDR) is associated with overproduction of Mr 170,000 membrane proteins (P-glycoproteins) caused by either gene amplification, transcriptional activation, or both. In rodents the amplified domain comprises genes that encode P-glycoproteins and at least five unrelated genes, one of which encodes the calcium-binding protein sorcin. The amplification and increased expression of these genes always includes one P-glycoprotein-encoding gene (pgp1 in hamsters, homologous to mdr1 in humans). In human MDR cells only elevated mdr1 expression has been shown thusfar, although another P-glycoprotein encoding gene (mdr3, homologous to hamster pgp3) is closely linked. Here we show that the human homolog of the hamster sorcin gene resides on chromosome 7 like the P-glycoprotein-encoding genes. Furthermore, gene classes designated 4, 5, and 6 are coamplified with mdr1 and mdr3 in the human ovarian carcinoma cell line 2780AD, which strongly suggests that the overall structure of the human MDR domain is the same as in rodents. Class 6 was moderately and mdr1 was highly overexpressed in this cell line. Four other human MDR cell lines also have much higher mdr1 overexpression than expected from the relatively low levels (2- to 30-fold) of gene amplification. This contrasts with the results of previous work with rodent MDR cells, in which the increase in P-glycoprotein mRNA levels usually parallels the increase in gene copy number. Although four of the five human MDR cell lines have coamplified mdr3, its expression was undetectable. Our results confirm the central role of the mdr1 (pgp1) gene in MDR and suggest that different cross-resistance patterns are not due to differential expression of different P-glycoprotein genes.

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We have found cDNAs corresponding to a novel human P-glycoprotein gene in liver cDNA banks. The sequence of the 3' part of this cDNA reveals a domainal organization of the derived protein similar to that of the known P-glycoproteins and an 80% amino acid homology with the product of the human mdr1 gene (Chen et al., 1986). The new gene lies within 500 kb from mdr1 as determined by pulsed field gradient gel electrophoresis and is designated mdr3, as it appears to correspond to the third of the three P-glycoprotein genes mapped in the hamster multidrug resistance domain. mdr3 yields a transcript of 4100 nucleotides, 400 nucleotides less than the mdr1 transcript; the difference is accounted for by the shorter 3'-untranslated region of the mdr3 mRNA. Our cDNAs provide evidence for alternative splicing of mdr3 pre-mRNAs. One alternative is an insert of seven amino acids between the two major blocks of the nucleotide binding site and another is a deletion of 43 or 47 amino acids covering the putative transmembrane segment 5a. We speculate that these alternatives superimposed on differential expression of P-glycoprotein homologues could provide an explanation for the large variation in cross-resistance patterns observed in cell lines selected for multidrug resistance with different cytostatic drugs.

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Pulsed field gradient electrophoresis allows the separation of large DNA molecules up to 2,000 kilobases (kb) in length and has the potential to close the resolution gap between standard electrophoresis of DNA molecules (smaller than 50 kb) and standard cytogenetics (larger than 2,000 kb). We have analysed the amplified DNA in four cell lines containing double minute chromosomes (DMs) and two lines containing homogeneously staining regions. The cells were immobilized in agarose blocks, lysed, deproteinized, and the liberated DNA was digested in situ with various restriction endonucleases. Following electrophoretic separation by pulsed field gel electrophoresis, the DNA in the gel was analysed by Southern blotting with appropriate probes for the amplified DNA. We find that the DNA in intact DMs is larger than 1,500 kb. Our results are also compatible with the notion that the DNA in DMs is circular, but this remains to be proven. The amplified segment of wild-type DNA covers more than 550 kb in all lines and possibly up to 2,500 kb in some. We confirm that the repeat unit is heterogeneous in some of the amplicons. In two cell lines, however, with low degrees of gene amplification, we find no evidence for heterogeneity of the repeats up to 750 (Y1-DM) and 800 kb (3T6-R50), respectively. We propose that amplicons start out long and homogeneous and that the heterogeneity in the repeat arises through truncation during further amplification events in which cells with shorter repeats have a selective advantage. Even if the repeats are heterogeneous, however, pulsed field gradient gels can be useful to establish linkage of genes over relatively short chromosomal distances (up to 1,000 kb). We discuss some of the promises and pitfalls of pulsed field gel electrophoresis in the analysis of amplified DNA.

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Multidrug-resistant cells are cross-resistant to a wide range of unrelated drugs, many of which are used in cancer chemotherapy. We constructed a cDNA library from RNA of the multidrug-resistant Chinese hamster ovary cell line CHRC5. By differential screening we isolated cDNAs derived from mRNAs that are overexpressed in this cell line. The cDNAs could be grouped in five classes on the basis of transcript lengths detected in RNA blots. We infer that each class codes for a separate protein. The corresponding genes are amplified 10 or 30 times in CHRC5 DNA, providing an explanation for the constitutive overexpression found in this cell line. Despite differential amplification, the genes may be linked in one large amplicon as indicated by the hybridization analysis of large fragments of CHRC5 DNA separated by pulsed field gradient gel electrophoresis. Therefore, some of these genes might be fortuitously coamplified and not contribute functionally to the resistant phenotype. It is also possible, however, that genes involved in drug resistance are clustered. One of our clones cross-hybridized with the recently described cDNA pCHP1 (J. R. Riordan, K. Deuchars, N. Kartner, N. Alon, J. Trent, and V. Ling, Nature [London] 316:817-819, 1985) encoding part of the 170-kilodalton P-glycoprotein, a protein which is frequently overproduced in multidrug-resistant cells. The nature of the four other genes is still unknown. Sequences of four of the five classes of cDNAs are conserved in mouse and human DNA.

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