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PubMed2003

Genotoxicity of pesticides: a review of human biomonitoring studies.

Pesticides constitute a heterogeneous category of chemicals specifically designed for the control of pests, weeds or plant diseases. Pesticides have been considered potential chemical mutagens: experimental data revealed that various agrochemical ingredients possess mutagenic properties inducing mutations, chromosomal alterations or DNA damage. Biological monitoring provides a useful tool to estimate the genetic risk deriving from an integrated exposure to a complex mixture of chemicals. Studies available in scientific literature have essentially focused on cytogenetic end-points to evaluate the potential genotoxicity of pesticides in occupationally exposed populations, including pesticide manufacturing workers, pesticide applicators, floriculturists and farm workers. A positive association between occupational exposure to complex pesticide mixtures and the presence of chromosomal aberrations (CA), sister-chromatid exchanges (SCE) and micronuclei (MN) has been detected in the majority of the studies, although a number of these failed to detect cytogenetic damage. Conflicting results from cytogenetic studies reflect the heterogeneity of the groups studied with regard to chemicals used and exposure conditions. Genetic damage associated with pesticides occurs in human populations subject to high exposure levels due to intensive use, misuse or failure of control measures. The majority of studies on cytogenetic biomarkers in pesticide-exposed workers have indicated some dose-dependent effects, with increasing duration or intensity of exposure. Chromosomal damage induced by pesticides appears to have been transient in acute or discontinuous exposure, but cumulative in continuous exposure to complex agrochemical mixtures. Data available at present on the effect of genetic polymorphism on susceptibility to pesticides does not allow any conclusion.

Chromosome Aberrations↗
PubMed2000

Inhibition of rat testicular androgenesis by a polychlorinated biphenyl mixture aroclor 1248.

Polychlorinated biphenyls (PCBs) are complex mixtures of congeners that exhibit carcinogenic and toxicant activities in a variety of mammalian tissues. Here, we studied the acute in vivo and in vitro effects of a commercially used PCB product, Aroclor 1248 (A1248), a mixture of tri-, tetra-, and pentachloro congeners. Single intraperitoneal (i.p.) or bilateral intratesticular (i.t.) injections of A1248 decreased serum androgen levels in both groups 24 h after injection. Chorionic gonadotropin-stimulated androgen production by acute testicular cultures from both groups was also reduced, and progesterone production was attenuated in cultures from i.t.-treated animals. The capacity of the postmitochondrial fractions from testes of i.t.-treated animals to convert pregnenolone to progesterone and progesterone to testosterone was reduced as well. In vitro studies revealed that a 10- to 15-min exposure of postmitochondrial testicular fractions and intact interstitial cells from normal animals to A1248 in a subnanomolar concentration range was sufficient to attenuate the conversion of pregnenolone to progesterone and progesterone to testosterone. At micromolar concentrations, A1248 added in vitro also inhibited the conversion of Delta(4)-androstendione to testosterone without affecting the viability of interstitial cells. These results indicate that A1248 down-regulates the testicular androgenesis by an acute inhibition of 3beta-hydroxysteroid dehydrogenase, 17alpha-hydroxylase/lyase, and 17beta-hydroxysteroid dehydrogenase activities.

17-Hydroxysteroid Dehydrogenases↗
PubMed1999

Effect of complex polyphenols and tannins from red wine (WCPT) on chemically induced oxidative DNA damage in the rat.

BACKGROUND: Flavonoids are polyphenolic antioxidants occurring in vegetables and fruits as well as beverages such as tea and wine which have been thought to influence oxidative damage. AIM OF THE STUDY: We wanted to verify whether a complex mixture of wine tannins (wine complex polyphenols and tannins, WCPT) prevent chemically-induced oxidative DNA damage in vivo. METHODS: Oxidative DNA damage was evaluated by measuring the ratio of 8-hydroxy-2'-deoxyguanosine (80HdG)/ 2-deoxyguanosine (2dG) x 10(-6) in hydrolyzed DNA using HPLC coupled with electrochemical and UV detectors. RESULTS: We treated rats with WCPT (57 mg/kg p.o.) for 14 d, a dose 10-fold higher than what a moderate wine drinker would be exposed to. WCPT administration significantly reduced the ratio of 80HdG/2dG x 10(-6) in liver DNA obtained from rats treated with 2-nitropropane (2NP) relative to controls administered 2NP only (33. 3 +/- 2.5 vs. 44.9 +/- 3.2 x 10(-6) 2dG; micro +/- SE; p<0.05). On the contrary, pretreatment with WCPT for 10 d did not protect the colon mucosa from oxidative DNA damage induced by 1, 2-dimethylhydrazine (DMH). 2NP and DMH are hepatic and colon carcinogens, respectively, capable of inducing oxidative DNA damage. CONCLUSIONS: WCPT have protective action against some types of chemically-induced oxidative DNA damage in vivo.

1,2-Dimethylhydrazine↗
PubMed1991

Studies on the mutagenicity of a zinc oxide-hexachloroethane smoke.

1. A suitable method has been developed for generating atmospheres of zinc oxide/hexachloroethane smoke (ZnHCE). 2. The smoke was investigated using the Ames test and the micronucleus assay. 3. It was weakly mutagenic to the bacteria, but in the bone marrow no increases in micronuclei were detected up to toxic levels of the smoke. 4. The method used here could be applied to other pyrotechnic mixtures which give rise to complex mixtures of products.

Animals↗
PubMedDate not supplied

Simultaneous synthesis of peptide libraries on single resin and continuous cellulose membrane supports: examples for the identification of protein, metal and DNA binding peptide mixtures.

Peptide libraries were simultaneously synthesized on single supports by double coupling 0.8 equivalents of an equimolar acylating amino acid mixture consisting of 19 amino acids (cysteine omitted) at randomized sites, thus compensating for the different coupling rates of the amino acids. Peptide epitope mixtures, as well as very complex mixtures such as a completely randomized hexapeptide, were prepared and analyzed by HPLC and amino acid analysis. The results obtained indicate that this method can be applied to the synthesis of peptide libraries. Parts of a simultaneously synthesized solution phase combinatorial library XXB1B2XX were successfully used for the detection of the linear epitope HFND of transforming growth factor-alpha (TGF alpha) recognized by the monoclonal antibody Tab2. Furthermore, novel combinatorial peptide libraries XXB1B2XX were prepared on continuous cellulose membrane supports, also allowing the identification of TGF alpha epitope sequences. In addition, peptide mixtures that bound to a double-stranded DNA (15mer) and silver were identified. These preliminary results indicate that cellulose-bound combinatorial peptide libraries can be used for the rapid and inexpensive screening of millions of peptides to identify single molecules that bind any given ligand such as proteins, nucleic acids and metals.

Amino Acid Sequence↗
PubMed1986

The separation of 99mTc(Sn)EHDP complexes by HPLC and GPC.

For the characterization of the multi-component bone-scan agent 99mTc(Sn)EHDP we have analysed the complex mixture with reversed phase ion pair chromatography (IPC) and soft gel permeation chromatography (GPC). With IPC five major complexes were found within a separation-time of 40 min. To avoid decomposition of the complexes during separation, the concentrations of EHDP and the reductant Sn(II) in the eluent had to be identical to the EHDP and Sn(II) concentrations used for the preparation of the complexes. To investigate the stability of the complexes we applied separation by IPC, followed by re-analysis of the fractions within 1 h and after 21-25 h. It appears that there is a state of equilibrium between the five complexes. Within 1 h after isolation the separated complexes were still more than 90% in their original form while after 20 h considerable amounts of the other complexes are found. The two complexes with the largest retention time with IPC are the most stable ones. When the total mixture was re-analysed after 26 h all five components appeared to be still present, but the relative amount of the most stable component had increased. Using GPC for the separation of the complex mixture, we found four major peaks within a separation time of 14 h. The elution orders of the complexes with the two separation methods are opposite.

Bone and Bones↗
PubMed2002

Cytological and biochemical effects of a mixture of 20 pollutants on isolated rainbow trout (Oncorhynchus mykiss) hepatocytes.

Isolated hepatocytes of rainbow trout (Oncorhynchus mykiss) were exposed to different concentrations (1x, 10x, 50x) of a complex mixture of 20 environmentally relevant contaminants (PAHs, PCBs, pesticides) typical of the chemical burden of surface waters and sediments of small rivers in southwest Germany to investigate sublethal cytological and biochemical alterations. Results document that all concentrations clearly induced biochemical and morphological changes. The activities of enzymes such as lactate dehydrogenase and catalase as well as the rate of lipid peroxidation were significantly increased, whereas acid phosphatase activity decreased. Cytopathological effects included deformation of cellular shape, deformation and dilation of the nuclear envelope, increase in heterochromatin, nuclear inclusions, heterogeneity of mitochondria, vesiculation of RER cisternae, as well as increases in myelinated bodies, lysosomes, and peroxisomes. If compared with cytological alterations induced in isolated rainbow trout hepatocytes after exposure to sediment extracts from rivers of corresponding contamination, cytological effects of exposure to the complex chemical mixture displayed numerous similarities.

Acid Phosphatase↗
PubMed1997

Genotoxicity assessment of suspended particulate matter in the Elbe river: comparison of Salmonella microsome test, arabinose resistance test, and umu-test.

This study evaluates the applicability of three bacterial short-term genotoxicity test systems to aquatic suspended particulate matter of the Elbe river. This material was sampled in sedimentation vessels after deposition periods of one month. It was extracted in a Soxhlet apparatus with toluene and methanol. Aqueous elutriates were prepared additionally. A solid phase method was developed that enables to incubate bacteria in contact with the particulate material. The test battery consists of two mutagenicity assays (the Ames-test and the Ara-test) and an SOS induction assay (the umu-test). Both mutagenicity assays came to nearly the same assessment of the samples of particulate matter of the Elbe. The quantitative response, however, was higher in the Ara-test. The particulate river material generally induced lower genotoxic potencies in the umu-test than in the mutagenicity assays. This lead to a completely different outcome of the umu-test; 29 out of 35 mutagenic samples were not SOS inducing. No quantitative or rank correlations between the concentrations of anthropogenic contaminants (PAHs, chlorinated hydrocarbons and metals) and the observed effects could be established on a 90% confidence limit. However, there is obvious correspondence between more contaminated regions of the river system and mutagenic effects (Ames- and Ara-test) in the samples from this region, as well as correspondence between low contaminated regions and the absence of mutagenicity. For this reason, the mutagenicity assays appear more favourable to describe the anthropogenic contamination with genotoxins in complex mixtures than the umu-test. The authors recommend the Ara-test for a first genotoxicity screening of complex environmental mixtures. This forward mutagenicity assay is advantageous due to higher effects and lower costs compared to the Ames-test. The development and use of a solid phase version of Ames- and Ara-test revealed the occurrence of a major part of particle-bound mutagens. The hydrophobic nature of these mutagens was also confirmed by the gradually decreasing effects with decreasing lipophilicity of the solvents. The results suggest that the solid phase test and the use of extracts complement each other in detecting mutagens of different lipophilicity. Both versions should be used in order to include a broad variety of compounds.

Arabinose↗
PubMed2006

Metal affinity capture tandem mass spectrometry for the selective detection of phosphopeptides.

We report a new method called metal affinity capture that when coupled with tandem mass spectrometry (MAC-MSMS) allows for the selective detection and identification of phosphopeptides in complex mixtures. Phosphopeptides are captured as ternary complexes with Ga(III) or Fe(III) and N(alpha),N(alpha)-bis(carboxymethyl)lysine (LysNTA) in solution and electrosprayed as doubly or triply charged positive ions. The gas-phase complexes uniformly dissociate to produce a common (LysNTA + H)+ ion that is used as a specific marker in precursor ion scans. The advantages of MAC-MSMS over the current methods of phosphopeptide detection are as follows. (1) MAC-MSMS uses metal complexes that self-assemble in solution at pH <5, which is favorable for the production of positive ions by electrospray. (2) Phosphorylation at tyrosine, serine, and threonine is detected by MAC-MSMS. (3) The phosphopeptide peaks in the mass spectra are encoded with the 69Ga-71Ga isotope pattern for selective recognition in mixtures. Detection by MAC-MSMS of singly and multiply phosphorylated peptides in tryptic digests is demonstrated at low-nanomolar protein concentrations.

Amino Acid Sequence↗
PubMed2005

1-DE MS and 2-D LC-MS analysis of the mouse bronchoalveolar lavage proteome.

Bronchoalveolar lavage fluid (BALF) is a complex mixture of proteins, which represents a unique clinically useful sampling of the lower respiratory tract. Many proteomic technologies can be used to characterize complex biological mixtures; however, it is not yet clear which technology(s) provide more information regarding the number of proteins identified and sequence coverage. In this study, we initially compared two common proteomic approaches, 2-D LC microESI MS/MS and 1-DE followed by gel slice digestion, peptide extraction and peptide identification by MS in characterization of the mouse BALF proteome; secondly, we identified 297 unique proteins from the mouse BALF proteome, greatly expanded the BALF proteome by about threefold regardless of species.

Amino Acid Sequence↗
PubMed2005

Functional screening of serine protease inhibitors in the medical leech Hirudo medicinalis monitored by intensity fading MALDI-TOF MS.

The blood-feeding invertebrates are a rich biological source of drugs and lead compounds to treat cardiovascular diseases because they have evolved highly efficient mechanisms to feed on their hosts by blocking blood coagulation. In this work, we focused our attention on the leech Hirudo medicinalis. We performed, by "intensity fading" MALDI-TOF mass spectrometry, a comprehensive detection and functional analysis of pre-existent peptides and small proteins with the capability of binding to trypsin-like proteases related to blood coagulation. Combining "intensity fading MS" and off-line LC prefractionation allowed us to detect more than 75 molecules present in the leech extract that interact specifically with a trypsin-like protease over a sample profile of nearly 2,000 different peptides/proteins in the 2-20-kDa range. Moreover we resolved 232 individual components from the complex mixture, 13 of which have high sequence homology with previously described serine protease inhibitors. Our findings indicate that such extracts are much more complex than expected. Additionally, intensity fading MS, when complemented with LC separation strategies, seems to be a useful tool to investigate complex biological samples, establishing a new bridge between profiling, functional peptidomics, and subsequent drug discovery.

Amino Acid Sequence↗
PubMed1994

Liquid chromatography/mass spectrometry of phospholipids using electrospray ionization.

An improved technique for phospholipid molecular species analysis was developed using high-performance liquid chromatography/mass spectrometry with the electrospray interface. Using the 0.5% ammonium hydroxide in a water-methanol-hexane mixture and a C-18 column, complex mixtures of phospholipid molecular species were separated and detected mainly as protonated or natriated molecular species. The response was linear over 2 orders of magnitude, allowing quantification of each molecular species. In comparison to the existing LC/MS techniques, marked improvement in sensitivity was observed. The present quantification limit is approximately 0.5 pmol before split (5 fmol after 1/100 split). The relative responses were more dependent on the head group identity rather than fatty acyl composition within a phospholipid class. In general, phosphatidylcholine (PC) species are most sensitively detected followed by phosphatidylethanolamine (PE) species. The sensitivity of phosphatidylserine (PS) in the positive ion mode is approximately 20 times less in comparison to PC under our condition.

Animals↗
PubMed2004

Complex organic chemical balms of Pharaonic animal mummies.

Millions of votive mummies of mammals, birds and reptiles were produced throughout ancient Egypt, with their popularity increasing during the reign of Amenhotep III (1400 bc) and thereafter. The scale of production has been taken to indicate that relatively little care and expense was involved in their preparation compared with human mummies. The accepted view is that animals were merely wrapped in coarse linen bandages and/or dipped in 'resin' before death. However, as with human mummification there was a range of qualities of treatments, and visual inspection of animal mummies suggests that the procedures used were often as complex as those used in humans (for example, evisceration and elaborate bandaging). Moreover, the ancient Egyptians treated animals with great respect, regarding them both as domestic pets and representatives of the gods; for example, the cat symbolized the goddess Bastet; the hawk, Horus; the ibis, Thoth, and so on. We report here the results of chemical investigations of tissues and wrappings from Pharaonic cat, hawk and ibis mummies using gas chromatography, gas chromatography-mass spectrometry, thermal desorption-gas chromatography-mass spectrometry and pyrolysis-gas chromatography-mass spectrometry. The analyses reveal the presence of highly complex mixtures of n-alkyl and cyclic biomarker components characteristic of fats, oils, beeswax, sugar gum, petroleum bitumen, and coniferous, Pistacia and possibly cedar resins. The mixture of balms is of comparable complexity to those used to mummify humans from the same period.

Animals↗
PubMed1992

Design, construction and application of a fully automated equimolar peptide mixture synthesizer.

A fully automated peptide synthesizer has been constructed that is capable of the synthesis of equimolar peptide mixtures and the simultaneous synthesis of 36 individual peptides. The synthesizer was constructed from a workstation of our own design utilizing a Zymark robot arm. A Macintosh II computer coordinates the movements of the robotic arm, the switching of over 40 solenoid valves and the monitoring of sensors in the workstation. The robot hands are used to deliver solvents from pressurized spigot lines and to pipet amino acid solutions from reservoirs to an array of reaction vessels. Liquid dispensing, reagent mixing and solvent removal are controlled from a multifunction I/O board in the computer. The design features of the synthesizer are presented, as well as the characterization of multiple individual peptides, a simple mixture of 19 components, and a complex mixture of 15,625 components.

Amino Acid Sequence↗
PubMed2001

Heritability of resistance to individual contaminants and to contaminant mixtures in the sheepshead minnow (Cyprindon variegatus).

Resistance heritability (the additive genetic variance out of the total phenotypic variance, signifying a population's potential to genetically adapt to detrimental levels of contamination) was quantified in the sheepshead minnow (Cyprinodon variegatus). Heritability was estimated for tolerance to individual contaminants (phenanthrene, zinc) and to contaminant mixtures (phenanthrene plus zinc, and a complex mixture with three metals and three polycyclic aromatic hydrocarbons). Estimates were obtained from resemblances between relatives, both parent-offspring pairs, and families of sibs and half-sibs. Heritabilities determined from parent-offspring regressions averaged only 0.08 (scale, 0-1), whereas resemblance among full sibs yielded heritabilities averaging 0.85. The half-sib analysis yielded heritabilities of -0.01 (sire component) and 0.77 (dam component). This pattern in the magnitude of heritabilities indicates that heritabilities for the resistance of C. variegatus to these chemicals are low (with the high resemblances among sibs being due to common environmental and dominance genetic variation rather than additive genetic variation). The parent-offspring regressions provide evidence that heritabilities may be lower if more contaminants are involved. Our results mean, then, that C. variegatus in contaminated environments is not likely to become resistant to these contaminants very rapidly, and that resistance may develop even more slowly as more contaminants become involved.

Adaptation, Physiological↗
PubMedDate not supplied

Optimization of enantiomeric separations in capillary electrophoresis by applying a design of experiments approach.

During early-phase pharmaceutical development, it is important to find an initial separation of enantiomeric compounds quickly in order to determine the enantiomeric purity of chiral drug substances. Highly selective screening methods are necessary to analyze the products to discover a satisfactory separation of the enantiomeric compounds. A screening approach based on the use of mixtures of multiple cyclodextrins in chiral capillary electrophoresis was employed to find the initial separation of chiral compounds. In a later phase of development, these complex methods need to be simplified for transferability. This study describes the simplification of the complex mixture of cyclodextrins into a single or dual system of only the enantioselective cyclodextrins. This was achieved by applying fractional factorial experimental designs to select the cyclodextrins that were responsible for the enantiomeric separation and response surface modeling designs for the optimization of the separation. In order to obtain robust methods, the concentration of the chiral selector, together with other important electrophoretic method parameters such as the concentration of the background electrolyte, pH, and run voltage, were optimized by employing a Box-Behnken experimental design.

Algorithms↗
PubMed1998

Analysis of recombinant human platelet-derived growth factor by reversed-charge capillary zone electrophoresis.

Reversed-charge capillary zone electrophoresis (RC-CZE) has been developed as a clipping (proteolysis) assay for homodimeric protein recombinant human platelet-derived growth factor (rhPDGF-BB), a major serum mitogenic factor involved in subcutaneous wound healing. When expressed in yeast, the protein is excreted as a fully folded homodimeric protein consisting of two antiparallel B chains held together by two interchain disulfide bonds. During fermentation, internal proteolysis (clipping between residues Arg32 and Thr33) and C-terminal truncation (Arg32 and Thr109) may occur. Internal proteolysis yields three potential forms of rhPDGF-BB: intact (both B chains are intact), single-clipped (one B chain is clipped), and double-clipped (both B chains are clipped). Clipping also creates new C-terminal sites for further C-terminal truncations and leads to a very complex mixture of isoforms. Routine baseline resolution of these three forms by various modes of HPLC proved unsuccessful. When the disulfide bonds of antiparallel chains are reduced, the complex peptide mixture can be analyzed by RP-HPLC; however, only the level of total clipping is identified. Since RC-CZE separation relies upon differences in molecular charge/size ratio, it can resolve the three rhPDGF-BB forms differing in the additional exposed residues. The choice of reversed-charge CZE columns (amine-coated column) allows proteins of high pI such as rhPDGF-BB (pI > 10) to be readily analyzed while minimizing protein loss from column adsorption. To simplify the electropherogram of clipped forms, the sample is treated first with carboxypeptidase B to reduce the charge microheterogeneity of partial Arg32 truncation. Analysis of rhPDGF-BB by RC-CZE yields a baseline separation between the three forms, intact and single- and double-clipped rhPDGF-BB.

Amino Acid Sequence↗
PubMed2004

Two-dimensional liquid chromatography-capillary zone electrophoresis-sheathless electrospray ionization-mass spectrometry: evaluation for peptide analysis and protein identification.

A peptide separation strategy that combines two-dimensional (2-D) liquid chromatography (LC)-capillary zone electrophoresis (CZE) with tandem mass spectrometry (MS/MS) is described for the identification of proteins in complex mixtures. To test the effectiveness of this strategy, a serum sample was depleted of the high-abundance proteins by methanol precipitation, digested with trypsin to generate a complex peptide mixture, and separated into 96 fractions by reversed-phase (RP)-LC. Compared to ion-exchange LC separations, RPLC provides much higher resolution and peak capacity. Fractions were collected off-line from the RPLC separation, and subjected to short 20 min CZE separations. The separated zones were introduced to the mass spectrometer through a sheathless electrospray ionization interface that is integrated on the separation capillary. The ease of fabrication of the interface and its durability allowed for the analysis of all fractions on a single capillary in a relatively short analysis time. A stable electrospray was produced at nanoliter flowrates by augmenting analyte electrophoretic and electroosmotic mobilities with pressure-assisted flow. Unlike first-dimensional ion-exchange LC fractionation, where there is a large degree of overlap, the CZE-MS results show less than 15% overlap between neighboring RPLC fractions.

Blood Proteins↗