DNA strand breakage, thymine glycol production, and hydroxyl radical generation induced by different samples of crystalline silica in vitro.

Five preparations of alpha-quartz [Min-U-Sil 5 (MQZ), MQZ pretreated with hydrofluoric acid (HFMQZ), Chinese standard alpha-quartz (CSQZ), and two German samples, DQ-12 and F600] and two preparations of the crystalline silica polymorphs, cristobalite and tridymite, previously characterized for surface area and surface charge, were evaluated for their relative activities in the following assays: (i) in vitro assays of short duration (< or = 15 min) for oxygen consumption and for generation of hydroxyl radicals (measured by electron spin resonance spin trapping), and (ii) in vitro assays of longer duration for DNA strand breakage (measured using linear DNA as a detector molecule) and for production of the oxidized DNA base, thymine glycol (measured by gas chromatography-mass spectrometry). Marked differences among the samples were found for their levels of oxygen consumption and of hydroxyl radicals' generation. All samples caused increased formation of thymine glycol, with wide variations in activity among samples. When normalized for equal surface area, the samples produced different levels of DNA strand breakage. Addition of hydrogen peroxide strongly accelerated DNA damage--more for cristobalite than for the alpha-quartz samples. DNA damage by quartz was enhanced by ferric chloride and inhibited by iron chelators. The order of relative activity of the samples varied with different types of in vitro assays and was not directly correlated to surface area. Electrophoretic mobility, as measured by zeta potential, was not significantly different among samples. The results suggest that the ability of different crystalline silica samples to generate a rapid burst of oxygen free radicals is distinct from their ability to induce DNA damage and DNA base oxidation over longer time periods. The relative activities of the samples in cellular assays (hemolysis of human erythrocytes; cytotoxicity and neoplastic transformation of BALB/3T3/A31-1-1 cells) were in turn markedly different from those listed above, suggesting a more critical role for surface area. The mechanisms of carcinogenesis by crystalline silica need to be further investigated in relation to the underlying physicochemical characteristics.

Direct interaction between crystalline silica and DNA - a proposed model for silica carcinogenesis.

Crystalline silica in aqueous buffer produced oxygen radicals that mediated in vitro DNA (deoxyribonucleic acid) strand breakage. The oxidized DNA base, thymine glycol, was also produced. The hydroxyl radical, responsible for most DNA damage, has a reaction distance of about 15 Angstroms, requiring close contact of silica with DNA. Fourier transform infrared spectroscopy of incubations of quartz particles with DNA showed distinct alterations in both DNA and quartz spectra and therefore indicated extensive hydrogen bonding between surface silanol groups and the phosphate-sugar backbone of DNA. Electron microscopy and energy dispersive X-ray spectroscopy of alveolar epithelial cells in fetal rat lung, exposed to quartz in culture, showed localization of quartz particles in the nuclei and mitotic spindles. Direct interaction of crystalline silica with DNA may be important in silica carcinogenesis by anchoring DNA close to sites of free radical production on the silica surface, or by interfering with DNA replication, repair, or the mitotic process.

Silica radical-induced DNA damage and lipid peroxidation.

In recent years, more attention has been given to the mechanism of disease induction caused by the surface properties of minerals. In this respect, specific research needs to be focused on the biologic interactions of oxygen radicals generated by mineral particles resulting in cell injury and DNA damage leading to fibrogenesis and carcinogenesis. In this investigation, we used electron spin resonance (ESR) and spin trapping to study oxygen radical generation from aqueous suspensions of freshly fractured crystalline silica. Hydroxyl radical (.OH), superoxide radical (O2.-) and singlet oxygen (1O2) were all detected. Superoxide dismutase (SOD) partially inhibited .OH yield, whereas catalase abolished .OH generation. H2O2 enhanced .OH generation while deferoxamine inhibited it, indicating that .OH is generated via a Haber-Weiss type reaction. These spin trapping measurements provide the first evidence that aqueous suspensions of silica particles generate O2.- and 1O2. Oxygen consumption measurements indicate that freshly fractured silica uses molecular oxygen to generate O2.- and 1O2. Electrophoretic assays of in vitro DNA strand breakages showed that freshly fractured silica induced DNA strand breakage, which was inhibited by catalase and enhanced by H2O2. In an argon atmosphere, DNA damage was suppressed, showing that molecular oxygen is required for the silica-induced DNA damage. Incubation of freshly fractured silica with linoleic acid generated linoleic acid-derived free radicals and caused dose-dependent lipid peroxidation as measured by ESR spin trapping and malondialdehyde formation. SOD, catalase, and sodium benzoate inhibited lipid peroxidation by 49, 52, and 75%, respectively, again showing the role of oxygen radicals in silica-induced lipid peroxidation.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanisms of carcinogenesis by crystalline silica in relation to oxygen radicals.

The carcinogenic effects of crystalline silica in rat lungs were extensively demonstrated by many experimental long-term studies, showing a marked predominance for adenocarcinomas originating from alveolar type II cells and associated with areas of pulmonary fibrosis (silicosis). In contrast with its effects in rats, silica did not induce alveolar type II hyperplasia and lung tumors in mice and hamsters, pointing to a critical role for host factors. Using these animal models, we are investigating the role of cytokines and other cellular mediators on the proliferation of alveolar type II cells. Immunohistochemical localization of TGF-beta 1 precursor in alveolar type II cells adjacent to silicotic granulomas was shown to occur in rats, but not in mice, and hamsters, suggesting a pathogenetic role for this regulatory growth factor. Recent investigations in our laboratory on the biologic mechanisms of crystalline silica included determination of anionic sites on crystalline silica surfaces by binding of the cationic dye Janus Green B; binding of crystalline silica to DNA, demonstrated by infrared spectrometry; production of oxygen radicals by crystalline silica in aqueous media; induction of DNA strand breakage and base oxidation in vitro and its potentiation by superoxide dismutase and by hydrogen peroxide; and induction by crystalline silica of neoplastic transformation and chromosomal damage in cells in culture. On the basis of these in vitro studies, we propose that DNA binding to crystalline silica surfaces may be important in silica carcinogenesis by anchoring DNA close to sites of oxygen radical production on the silica surface, so that the oxygen radicals are produced within a few A from their target DNA nucleotides.

DNA binding to crystalline silica characterized by Fourier-transform infrared spectroscopy.

The interaction of DNA with crystalline silica in buffered aqueous solutions at physiologic pH has been investigated by Fourier-transform infrared spectroscopy (FT-IR). In aqueous buffer, significant changes occur in the spectra of DNA and silica upon coincubation, suggesting that a DNA-silica complex forms as silica interacts with DNA. As compared to the spectrum of silica alone, the changes in the FT-IR spectrum of silica in the DNA-silica complex are consistent with an Si-O bond perturbation on the surface of the silica crystal. DNA remains in a B-form conformation in the DNA-silica complex. The most prominent changes in the DNA spectrum occur in the 1225 to 1000 cm-1 region. Upon binding, the PO2- asymmetric stretch at 1225 cm-1 is increased in intensity and slightly shifted to lower frequencies; the PO2- symmetric stretch at 1086 cm-1 is markedly increased in intensity and the band at 1053 cm-1, representing either the phosphodiester or the C-O stretch of DNA backbone, is significantly reduced in intensity. In D2O buffer, the DNA spectrum reveals a marked increase in intensity of the peak at 1086 cm-1 and a progressive decrease in intensity of the peak at 1053 cm-1 when DNA is exposed to increasing concentrations of silica. The carbonyl band at 1688 cm-1 diminishes and shifts to slightly lower frequencies with increasing concentrations of silica. The present study demonstrates that crystalline silica binds to the phosphate-sugar backbone of DNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Generation of thiyl and ascorbyl radicals in the reaction of peroxynitrite with thiols and ascorbate at physiological pH.

Electron spin resonance (ESR) spin trapping was utilized to investigate the reaction of peroxynitrite with thiols and ascorbate at physiological pH. The spin trap used was 5,5-dimethyl-1-pyrroline N-oxide (DMPO). The reaction of peroxynitrite with DMPO generated 5,5-dimethylpyrrolidone-(2)-oxy-(1) (DMPOX). Formate enhanced the peroxynitrite decomposition but did not generate any detectable amount of formate-derived free radicals. Thus, the spin trapping measurements provided no evidence for hydroxyl (.OH) radical generation in peroxynitrite decomposition at physiological pH. Thiols (glutathione, cysteine, and penicillamine) and ascorbate reacted with peroxynitrite to generate the corresponding thiyl and ascorbyl radicals. The one-electron oxidation of thiols by peroxynitrite may be one of the important mechanisms for peroxynitrite-induced toxicity and ascorbate may provide a detoxification pathway.

Binding of the cationic dye, Janus green B, as a measure of the specific surface area of crystalline silica in aqueous suspension.

Twelve preparations of crystalline silica, with a wide range of particle sizes, were assayed by a new method, which measures surface adsorption of the cationic dye Janus green B to crystalline silica samples in a buffered aqueous suspension. The same samples were also assayed for total surface area by the Brunauer-Emmett-Teller (BET) method of surface adsorption of nitrogen gas. A strong linear correlation was found between the two methods of measurement (r = 0.977). Reproducible specific surface area measurements by the Janus green B adsorption method were made on 2-mg samples using ordinary visible wave-length spectrophotometric equipment, whereas the BET method necessitated sample sizes in excess of 100 mg and more specialized instrumentation. Five size-fractionated preparations from the same Min-U-Sil alpha-quartz sample showed an increase in BET surface area and Janus green B binding per unit weight with decreasing particle size. Among four standard alpha-quartz samples tested, Min-U-Sil 5 and F600 had the lowest specific surface areas, whereas DQ-12 and Chinese standard alpha-quartz had much higher surface areas. The synthetic silica preparations cristobalite and tridymite had intermediate surface areas. Binding by the cationic dye Janus green B is consistent with a surface charge mechanism and provides a useful new technique for the assessment of surface characteristics of crystalline silica samples. Its linear relationship to surface area suggests that the ratio of aqueous surface charge to surface area is constant for different crystalline silica preparations. Comparison of surface areas for different preparations of crystalline silica is important in understanding the relative activities of these preparations in studies on mechanisms of silicosis and silica-induced lung cancer.

Oxidative DNA damage by crystalline silica.

Using a simple DNA strand breakage assay, we detected the production of oxidant species, probably hydroxyl free radicals, in buffered suspensions of crystalline silica at pH 7.4. DNA damage was affected by the presence of oxygen and was accelerated by superoxide dismutase and by hydrogen peroxide. Deferoxamine blocked damage by hydrogen peroxide and silica but accelerated DNA damage by silica alone and by superoxide dismutase and silica. DNA damage was blocked by catalase and by the scavenging agents dimethyl sulfoxide and sodium benzoate. Chemical etching of crystalline silica to remove impurities by treatment of the surface with hydrofluoric acid resulted in markedly diminished DNA damaging ability. Even preparations of crystalline silica previously characterized as highly pure contained trace iron impurities in amounts significant enough to produce oxygen free radicals in aqueous suspension. Both superoxide and Fenton reaction oxidants were produced. We conclude that silica is able to mediate DNA strand breakage in vitro and that this DNA damage may be an important factor in silica toxicity.

Von Hippel-Lindau (VHL) disease: distinct phenotypes suggest more than one mutant allele at the VHL locus.

As part of an attempt to locate the von Hippel-Lindau locus (VHL) on chromosome 3, we evaluated 41 families with von Hippel-Lindau disease from the United States and Canada. One large family was identified whose disease phenotype was distinct from typical VHL. The most common disease manifestation was pheochromocytoma occurring in 57% (27/47) of affected family members. Few (4/47) affected family members had symptomatic spinal or cerebellar hemangioblastomas; no affected family member had renal cell carcinoma (0/47) or pancreatic cysts (0/24). Previously, genetic analysis demonstrated that the disease manifestations in this family were linked to RAF1 and D3S18, markers shown to be linked to typical VHL. These results suggest that there are mutant alleles at the VHL locus associated with distinct tissue specificities.

Isolation and regional localization of a large collection (2,000) of single-copy DNA fragments on human chromosome 3 for mapping and cloning tumor suppressor genes.

A collection of 2,000 lambda phage-carrying human single-copy inserts (greater than 700 bp) were isolated from two chromosome-3 flow-sorted libraries. The single-copy DNA fragments were first sorted into 3p and 3q locations and about 700 3p fragments were regionally mapped using a deletion mapping panel comprised of two human-hamster and two-human-mouse cell hybrids, each containing a chromosome 3 with different deletions in the short arm. The hybrids were extensively mapped with a set of standard 3p markers physically localized or ordered by linkage. The deletion mapping panel divided the short arm into five distinct subregions (A-E). The 3p fragments were distributed on 3p regions as follows: region A, 26%; B, 31%; C, 4%; D, 4% and E, 35%. We screened 300 single-copy DNA fragments from the distal part of 3p (regions A and B) with ten restriction endonucleases for their ability to detect restriction fragment length polymorphisms (RFLPs). Of these fragments 110 (36%) were found to detect useful RFLPs; 35% detected polymorphisms with frequency of heterozygosity of 40% or higher, and 25% with frequency of 30% or higher. All polymorphisms originated from single loci and most of them were of the base pair substitution type. These RFLP markers make it possible to construct a fine linkage map that will span the distal part of chromosome 3p and encompasses the von Hippel-Lindau disease locus. The large number of single-copy fragments (2,000) spaced every 100-150 kb on chromosome 3 will make a significant contribution to mapping and sequencing the entire chromosome 3. The 300 conserved chromosome 3 probes will increase the existing knowledge of man-mouse homologies.

Genetic changes in human adrenocortical carcinomas.

Recent studies have suggested that loss of heterozygosity at loci on the short arm of human chromosome 11 (11p) may be important in the pathogenesis of benign and malignant adrenal cortical tumors. To test this concept, adrenocortical carcinomas from nine patients and benign adrenal cortical lesions from eight patients were tested for loss of alleles at loci on human chromosomes 11, 13, and 17. All patients with adrenocortical carcinoma whose normal somatic tissues were heterozygous for a locus on chromosome 17p had lost alleles in the tumor. Four of six patients with adrenocortical carcinoma who were heterozygous for one or more alleles on chromosome 11p in normal tissues had lost 11p alleles in the tumor. Three of six patients with adrenocortical carcinoma showed loss of 13q alleles in the tumor. Loss of alleles on chromosomes 11p, 13q, and 17p was observed in primary tumors and metastases but not in adrenocortical adenomas or hyperplastic lesions of the adrenal cortex. One patient with adrenocortical carcinoma had a somatic mutation in the HRAS1 gene in the normal adrenal gland. The consistency of the genetic changes on chromosomes 11p, 13q, and 17p suggests that they are important in the pathogenesis of adrenocortical carcinoma.

Isolation and characterization of relaxin from the sand tiger shark (Odontaspis taurus).

A peptide with relaxin activity in guinea pigs but not in mice has been extracted from the ovaries of pregnant sand tiger sharks (Odontaspis taurus). The structural similarity of this peptide to porcine relaxin includes molecular size approximately 6000 daltons), number of chains, and possibly, disulfide cross-links. The relaxin-type peptide isolated from shark ovaries contains the amino acid residues tyrosine, proline, and histidine, which are absent in the porcine hormone. The amino acid composition of shark relaxin, therefore, resembles that of porcine insulin to a greater extent than does the amino acid composition of porcine relaxin. This finding supports the idea that shark relaxin may be a primitive relaxin that has undergone fewer mutations than porcine relaxin since the putative duplication of the insulin gene. The data presented here suggest that the putative duplication of the insulin gene, which might have given rise to relaxin, has occurred much earlier than the separation of sharks from the general branch of animals that eventually gave rise to mammals.