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M Vanderlaan

Publications and source records attributed to M Vanderlaan.

At least 19 recordsLinked to original sources
PubMed1995

Perfusion immunoassay for acetylcholinesterase: analyte detection based on intrinsic activity.

A quantitative, two-column, HPLC-based assay requiring only 30 min to complete is reported. Amniotic fluid proteins are first fractionated on a size-exclusion column; the fraction containing the M(r) 280,000 neural acetylcholinesterase (AChE) is then diverted to a second column, an ImmunoDetection cartridge derivatized with an anti-AChE antibody. The immobilized antibody traps the enzyme, then substrate is flowed through the cartridge and the product is detected. For a positive result, the enzyme must have a molecular mass corresponding to the neural-AChE, be recognized by the antibody and be active in converting the substrate into product. The assay is sensitive in the clinically relevant range. The method provides rapid quantitative analysis using an automated instrument projected to be suitable for screening large numbers of samples.

Acetylcholinesterase↗
PubMed1993

Effects of diet and folate on levels of micronucleated polychromatic erythrocytes.

We describe a study examining the effects of fried beef consumption on the frequency of micronuclei in RNA-positive polychromatic erythrocytes (PCEs) in humans. 5 splenectomized individuals participated in a 2-week experiment consisting of 3 phases. During the first and last phases the subjects refrained from eating cooked meat for 4-5 days. This was designed to clear their system of mutagens found in cooked-meat products. In the second phase each person ate a similar diet, but also consumed 4 quarter-pound well-done hamburgers per day for 4 days. Erythrocyte-folate levels were also measured for each donor. No association between diet phase and micronucleus frequencies was observed among the 2 subjects with normal levels of folate. However, among the 3 low-folate donors, the frequency of micronucleated PCEs appeared to be associated with diet. Micronucleus frequencies began to increase 1 day following onset of the beef phase, and started to decline 1 day after finishing this phase. These observations are in agreement with erythrocyte maturation kinetics following short-term exposure. A repeat study performed on one of the low-folate donors consisted of two beef phases separated by a vegetarian phase. Beef in one phase was fried (high in mutagenic amino-imidazoazaarenes [AIAs]) while the beef in the other phase was fried after first being microwave-cooked (low in AIAs). Significant increases in the micronucleus frequency were not observed in this experiment, suggesting that AIAs formed during frying were not solely responsible for the induction of micronuclei.

Adult↗
PubMed1991

The effect of dose and enzyme inducers on the metabolism of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in rats.

Male Fischer 344 rats were given a single dose of 0.03-30 mg/kg of [2-14C]2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine ([14C]PhIP), the radioactivity in urine and feces was determined over 48 h, and the major metabolites were identified and quantified. Dose had little effect on the profile of metabolites in the urine but did influence the profile in the feces. PhIP was more efficiently metabolized at higher doses. In addition, rats were pretreated with Aroclor 1254 (PCB), 3-methylcholanthrene (MC), phenobarbital (PB), PhIP and corn oil prior to a single dose of [14C]PhIP, and compared with a control group receiving [14C]PhIP only. The major metabolites in the urine and feces were quantitated for each group, as well as PhIP binding to serum proteins, hemoglobin and selected tissues. Pretreatment with MC and PCB resulted in an increase in the amount of 4'-hydroxylation of PhIP and a decrease in the amount of N-hydroxylated metabolites in the urine. Pretreatment with PB resulted in an increase in the amount of N-hydroxylated metabolites, but a decrease in 4'-hydroxylation. Pretreatment with either MC or PCB resulted in an increase in PhIP binding to the liver and kidney, while reducing the binding in other tissues. Animals pretreated with PhIP showed few significant differences from the untreated group, while pretreatment with PB in general resulted in a decrease of PhIP binding in tissues.

Animals↗
PubMed1991

Immunochemical detection of rodent hepatic and urinary metabolites of cooking-induced food mutagens.

Monoclonal antibodies, previously selected to bind either 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) or 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) were evaluated to determine their binding properties with several known metabolites of these compounds purified from rat hepatocyte cultures. Both 2-N- and 5-substituted MeIQx metabolites were recognized by antibodies AIA-2 and AIA-11, while antibodies AIA-1 and AIA-12 bound N-substituted metabolites only. Anti-PhIP antibodies bound N-hydroxy-PhIP, N-sulfinamide-glutathione-PhIP and N-hydroxy-glucuronide-PhIP. Immunoaffinity columns incorporating these antibodies were used to concentrate and purify both the unmetabolized parent compounds and several relatively non-polar metabolites from the urine of rats exposed either to MeIQx or PhIP. Several of these metabolites corresponded with available standards of previously identified polar conjugate metabolites, e.g. N-sulfamate-MeIQx and N(OH)-gluc-PhIP, while others are as yet unidentified. Immunoaffinity chromatography is demonstrated as a promising means of selectively concentrating metabolites of PhIP and MeIQx from urine.

Animals↗
PubMed1991

Azido- and nitro-PhIP, relatives of the heterocyclic arylamine and food mutagen PhIP--mechanism of their mutagenicity in Salmonella.

Azido-PhIP (2-azido-1-methyl-6-phenylimidazo[4,5-b]pyridine) and nitro-PhIP (2-nitro-1-methyl-6-phenylimidazo[4,5-b]pyridine) have been synthesized and characterized chemically. The mutagenic potencies of azido-PhIP (with photoactivation by near UV irradiation), of nitro-PhIP (without exogenous activation) and of the heterocyclic amine PhIP (with activation by rat liver S9) were evaluated by means of the reversion assay in Salmonella typhimurium. Like PhIP, azido- and nitro-PhIP were potent mutagens in the strain TA98. In addition to TA98, the strains YG1024 with increased and TA98/1,8-DNP6 deficient in acetyltransferase activity and TA98NR deficient in nitroreductase were used. Photolysis of azido-PhIP generates a very short-lived mutagen whose mutagenic potency is similar in all strains used and therefore not dependent on the acetyltransferase and nitroreductase. These findings are analogous to previous ones with azidofluorene and suggest that a short-lived DNA-binding arylnitrenium ion is formed directly by the photolysis of azido-PhIP. The nitroreductase contributes to the mutagenic potency of nitro-PhIP; in TA98NR it is 37% of that in TA98. The acetylator status of the strains influences the mutagenic potencies of PhIP and nitro-PhIP only weakly. This contrasts with findings obtained with 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and nitro-IQ and with other amino- and nitro-imidazoarenes which lose most of their mutagenic activity in the acetyltransferase-deficient strain. This atypical behavior of PhIP suggests that the presumed metabolite, N-hydroxy-PhIP, differs from the N-hydroxy metabolites of other heterocyclic amino- and nitro-imidazoarenes in that in Salmonella it is not significantly activated by O-acetylation. It must therefore either react directly with DNA or be activated in a different way. This divergent behavior of PhIP and N-hydroxy-PhIP in Salmonella may also be a key to the understanding of several 'unusual' properties of PhIP in mammalian cells and organisms.

Biotransformation↗
PubMed1990

Flow cytometric analysis of erythrocyte populations in Tn syndrome blood using monoclonal antibodies to glycophorin A and the Tn antigen.

Flow cytometric analysis employing monoclonal antibodies to the Tn antigen and glycophorin A was used to characterize the erythrocyte populations present in blood samples from individuals with Tn syndrome. Four monoclonal antibodies specific for the Tn antigen, Gal-NAc monosaccharide, on human erythrocytes were obtained from a fusion of splenocytes from a Biozzi mouse immunized with red cells from a Tn individual. These monoclonal antibodies specifically recognize GalNAc monosaccharide sites located on the erythrocyte cell surface sialoglycoproteins, glycophorin A and glycophorin B, and do not bind to fixed normal red cells presenting the Neu-NAc alpha 2-3Gal beta 1-3(NeuNAc alpha 2-6)GalNAc alpha 1-O-Ser(Thr) tetrasaccharide or to fixed neuraminidase-digested cells presenting the Gal-GalNAc disaccharide. The percentages of Tn-positive red cells in samples from six unrelated Tn donors ranged from 28 to 99%. Binding of the glycophorin A-specific monoclonal antibodies showed that the erythrocytes composing the Tn-negative fraction presented normal amounts of the M and N epitopes on glycophorin A. The presumed somatic mutational origin of Tn-positive cells was tested in blood samples from five normal donors; three possible Tn cells were observed after analysis of a total of 1.1 x 10(7) erythrocytes, suggesting that the frequency of such cells in normal individuals is less than 1 x 10(-6).

Antibodies, Monoclonal↗
PubMed1990

Role of metabolism on the DNA binding of MeIQx in mice and bacteria.

We report the effects of several inducers of P450 metabolizing enzymes on DNA adduct formation by 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) in C57BL/6 mice. We also examined the role of N:O-acetylation and the nitrenium ion in the genotoxicity of MeIQx, since these have been implicated in the activation of other aminoimidazoazaarenes (AIA) to DNA reactive species. Mice were given phenobarbital (PB), Aroclor 1254, beta-naphthoflavone (BNF) or corn oil, i.p., followed 3-5 days later with oral administration of MeIQx. Induction of Aroclor and BNF produced DNA with 8-fold more adducts than either the corn oil-alone or PB-treated animals. Both corn oil-alone and PB-treated animals were similar. Four major adducts were found in all cases with no differences among inducers as judged by co-chromatography. Azido-MeIQx induced calf-thymus-DNA adducts produced identical adduct profiles to those seen for the mouse DNA. Similar adduct profiles were obtained from Salmonella TA98, and the nitroreductase deficient strains (TA98NR and TA98/1,8-DNP6) exposed to MeIQx in the presence of Aroclor-induced-mouse-liver S9. Adduct frequencies in TA98/1,8-DNP6 were significantly lower than in TA98 and TA98NR. The data described in this report demonstrate that induction quantitatively increases adduct numbers but does not affect the types of DNA damage. These data also suggest that the same DNA reactive intermediates are formed in vivo as in vitro and support the hypothesis that the metabolism of MeIQx involves the P450I family of isozymes, N:O-acetyltransferases and possibly a nitrenium ion. The application of radioanalytic scanners for quantitation of 32P-postlabelling adduct maps is described.

Animals↗
PubMed1990

Cell kinetics of rat 9L brain tumors determined by double labeling with iodo- and bromodeoxyuridine.

Rats with 9L brain tumors received intraperitoneal injections of iododeoxyuridine (IUdR) and bromodeoxyuridine (BUdR) to estimate the duration of the deoxyribonucleic acid (DNA) synthesis phase (Ts) and the potential doubling time (Tp) of individual tumors. Different sequences and intervals (2 or 3 hours) of IUdR and BUdR administration were evaluated. After denaturation, tumor sections were reacted with Br-3, a monoclonal antibody that identifies only BUdR, and then were stained immunohistochemically by the avidinbiotin complex method. An antibody that recognizes both IUdR and BUdR, IU-4, was applied to the sections and identified by the alkaline phosphatase-antialkaline phosphatase method. Nuclei labeled only with IUdR stained blue, while those labeled with BUdR or with BUdR and IUdR stained brown. The fraction of cells that either left or entered the S-phase during the time between administration of IUdR and BUdR was measured to calculate Ts and Tp, assuming that the labeled cohort completed the DNA synthesis at a constant rate. The Ts was 8.8 hours (coefficient of variation (cv) = 0.05) and the Tp was 64.2 hours (cv = 0.08). The sequence and interval of administration of IUdR and BUdR had a minimal effect on Ts and Tp. In studies of 9L cells in monolayer culture, the Ts was 9.6 hours (cv = 0.08) and the TP was 30.6 hours (cv = 0.06). Double labeling with BUdR and IUdR allows the duration of the S-phase and potential doubling time of individual brain tumors to be estimated in situ from a single biopsy specimen.

Animals↗
PubMed1989

Monoclonal antibodies to 2-amino-1-methyl-6-phenylimidazo[4,5-b] pyridine (PhIP) and their use in the analysis of well-done fried beef.

Four monoclonal antibodies, PhIP-1, -2, -3 and -4, have been produced that bind to PhIP (2-amino-1-methyl-6-phenyl-imidazo[4,5-b]pyridine), a mutagenic aromatic amine produced in meat by cooking. The immunogen was an analog of PhIP conjugated to a carrier protein through the 2-amino group of PhIP, leaving the pyridine and phenyl rings unmodified. The antibodies show a range of binding specificities for PhIP and its structural analogs, with PhIP-1 being the most highly selective, requiring the entire PhIP molecule with the exception of the exocylic amino group to be present for recognition. A competition enzyme linked immunosorbent assay (ELISA) incorporating these antibodies was developed and applied to the analysis of fried meat extracts differing in mutagen content. In extracts of fried beef, more PhIP-like material was found than would be expected based on chemical estimates of the amount of PhIP, suggesting that frying produces other compounds that are structurally similar to PhIP. These PhIP-like compounds were separated from PhIP using HPLC. Addition of creatinine to the beef prior to frying increased both the mutagenicity of the extracts and the amount of PhIP-like material. Microwave pre-treatment prior to frying reduced both immunochemical reactivity and mutagenicity.

Antibodies, Monoclonal↗
PubMed1989

Double labeling with iodo- and bromodeoxyuridine for cell kinetics studies.

The rate of progression through the cell cycle was determined in five human glioma cell lines by a new sequential immunohistochemical staining technique. The cells were labeled first with iododeoxyuridine (IdUrd) for 1-3 hr and then with bromodeoxyuridine (BrdUrd) for 30 min. Labeled cells were identified with Br-3, a monoclonal antibody that recognizes only BrdUrd, and with IU-4, an antibody that recognizes both IdUrd and BrdUrd. Each slide was stained sequentially, first with the immunoperoxidase method for Br-3 and then with the alkaline phosphatase-anti-alkaline phosphatase method for IU-4. Cells that were positive only for IU-4 represented the fraction of S-phase cells that passed into the G2 phase during the period of incubation with IdUrd. The rates of progression measured by this method were constant in each cell line and resulted in smaller standard errors than were obtained by measurements from specimens stained singly for IdUrd and BrdUrd in different slides. The duration of the S-phase calculated from this fraction in the five cell lines ranged from 8-13 hr; the estimated potential doubling times were 25-32 hr and were very similar to the actual doubling times.

Bromodeoxyuridine↗
PubMed1988

Monoclonal antibodies for the immunoassay of mutagenic compounds produced by cooking beef.

A family of 2-amino-N-methylimidazoazaarene (AIA) mutagens are produced in meats by cooking, and are of concern as potential carcinogens in the human diet. These are potent genotoxins in bacterial mutation assays, but are present only in trace quantities in cooked foods. Conventional analytical chemistry methods have allowed us to identify six members of the AIA class, but these methods are much too labor-intensive for us to quantify mutagens in large numbers of meat samples. To improve the assay of these mutagens we have developed six monoclonal antibodies (named IQ-1, IQ-2, AIA-1, AIA-2, AIA-4, and AIA-7) and demonstrated their utility in an immunoassay for cooked food mutagens. Each antibody has its own unique binding selectivity pattern; some are compound-specific, and some class-specific. A comparison was made between cooked beef extracts that differed 200-fold in mutagenic activity. The high-mutagen extract had significantly more material that was immunologically cross-reactive with the anti-AIA antibodies than did the low-mutagen extract. Taken as a set, these antibodies will allow rapid quantitation of foods for several AIA mutagens, and will aid in the isolation and characterization of other AIAs and AIA-metabolites.

Animals↗
PubMed1987

Monoclonal antibodies for dioxin: antibody characterization and assay development.

A set of 5 anti-dioxin monoclonal antibodies (mAbs), named DD-1, DD-3, DD-4, DD-5 and DD-6, have been isolated. In order to evaluate the ability of these mAbs to recognize various kinds of polychlorinated dibenzodioxins and dibenzofurans, a competition enzyme-linked immunosorbtion assay (ELISA) was developed. All 5 antibodies recognize tetrachloro- and pentachloro-dibenzodioxins and -dibenzofurans. They fail to bind either non-chlorinated, mono-, hexa-, or octa-chlorinated dibenzodioxins, nor do they recognize non-chlorinated, octachloro- or 1,2,3,4,8,9-hexachloro-dibenzofurans. Chlorine substitution on both rings appears necessary for antibody recognition. In the course of our experiments, 3 of the mAbs did not recognize any of the polychlorinated biphenyls (PCBs) tested, while 2 mAbs (DD-1 and DD-6) weakly recognized the 3,3',4,4'-tetrachloro congener. DD-4 and DD-5 are the most specific of the antibodies for the dibenzodioxin and dibenzofuran structure. They do not recognize any of a panel of chlorinated phenols, benzenes, or pesticides. Significantly, these antibodies do not react with PCBs, pentachlorophenol, 2,4-dichlorophenoxyacetic acid, trichlorophenol, or 2,4,5-trichlorophenoxyacetic acid (the latter is weakly recognized by DD-6), any or all of which might be present in large quantities in some dioxin-contaminated samples. Finally, the competition ELISA is able to easily detect 0.5 ng of the most toxic dioxin congener, 2,3,7,8-tetrachlorodibenzo-p-dioxin. It should thus prove useful as an environmental screen for contamination.

Animals↗
PubMed1986

Monoclonal antibodies recognizing single amino acid substitutions in hemoglobin.

Four monoclonal antibodies (mAb) to non-human primate hemoglobin referred to as Cap-4, Cap-5, Rh-2, and Rh-4, and two mAb to human hemoglobin, referred to as H-1 and H-3 were isolated and were partially characterized. Binding studies with these mAb on a panel of hemoglobins and isolated alpha and beta globin chains revealed a unique reactivity pattern for each mAb. Amino acid sequence analysis of the antigens used to generate the binding data suggests that the specific recognition of certain hemoglobin antigens by each mAb is controlled by the presence of a particular amino acid at a specific position within the epitope. The use of synthetic peptides as antigens confirmed this observation for five of the mAb. No synthetic peptides were tested with the sixth mAb, Rh-2. The amino acids required for binding of mAb Cap-4, Cap-5, Rh-4, and Rh-2 to hemoglobin are alanine at beta 5, threonine at beta 13, glutamine at beta 125, and leucine at alpha 68. The non-human primate hemoglobin antibodies require a specific amino acid that is not present in human hemoglobin. The amino acid required for binding of Cap-4, Cap-5, and Rh-4 could arise by a single base change in the beta globin gene, whereas the amino acid required for Rh-2 binding would only occur if two base changes occurred. Thus these mAb are candidate probes for a somatic cell mutation assay on the basis of the detection of peripheral blood red cells that possess single amino acid substituted hemoglobin as a result of single base substitutions in the globin genes of precursor cells.

Amino Acid Sequence↗
PubMed1986

Improved high-affinity monoclonal antibody to iododeoxyuridine.

Three monoclonal antibodies (Mabs), IU-1, IU-4, and B-44 were evaluated in enzyme immunoassays (ELISA) and by flow cytometry for their abilities to recognize bromodeoxyuridine (BrdUrd)- and iododeoxyuridine (IdUrd)-substituted DNA's, nucleotides, and nucleosides. IU-4 is a new Mab, derived from mice immunized with 5-iodo-2'-deoxyuridine-5'-monophosphate (IdUMP) conjugated (IdUMP) conjugated through the phosphate group to albumin. This immunogen was selected to resemble IdUMP in DNA. In competition ELISA assays, IU-4 prefers IdUrd to BrdUrd and prefers halogenated nucleotides over the corresponding nucleosides. In both ELISA and flow analysis, IU-4 recognizes IdUrd in DNA at substitution frequencies at least as low as one IdUrd one per 1,000 normal bases. The high affinity of IU-4 for IdUrd-DNA contrasts with IU-1 and B-44, which show a strong binding dependency on the frequency of base substitution and require DNA that is essentially fully substituted with BrdUrd for binding in both flow and ELISA assays. The high affinity of IU-4 for IdUrd in DNA and its independence of IdUrd residue spacing make it a superior reagent for the quantitative labeling of halogenated thymidine analogues in whole cells.

Animals↗
PubMed1986

Expression of proliferating cell nuclear antigen (PCNA)/cyclin during the cell cycle.

The expression of proliferating cell nuclear antigen (PCNA), also called cyclin, was quantified in the cell lines SP2/0 and MOLT-4 and in mouse splenocytes induced to proliferate in vitro with mitogens. Autoantibody from a patient with systemic lupus erythematosus was used to label PCNA in cell suspensions after the cells had been fixed and permeabilized. In the same cells DNA was stained by propidium iodide. The cells were then analysed by flow cytometry for PCNA and DNA content. The PCNA profiles in proliferating spleen cells and the cell lines were similar. Most G0-G1 cells did not express significant amount of PCNA. A dramatic increase in PCNA immunofluorescence was observed in late G1 cells, and further increases were observed in S-phase cells. G2-M cells showed a reduced level of PCNA immunofluorescence relative to S-phase cells but were still elevated relative to G0-G1 cells. Proliferating cells arrested at the G1-S boundary by exposure to cytosine arabinoside showed an increased PCNA immunofluorescence as compared to unstimulated cells.

Animals↗