The refractory period of the audible "crack" after lumbar manipulation: a preliminary study.

OBJECTIVE: This study evaluates if side posture lumbar manipulation is associated with a refractory period of the audible "crack" and if so, to quantify this refractory period across subjects. METHODS: Three subjects were exposed to multiple "baseline" side posture manipulations until no further audible cracks were recorded. "Test-refractory period" manipulations were administered after a set time (ie, potential refractory period) at which point the number of audible cracks was recorded. The refractory period was declared when a minimum of 50% of the baseline audible "cracks" had recovered during the test manipulations. The study design included 2 clinicians who performed side posture lumbar manipulation on asymptomatic subjects ranging from 38 to 49 years of age. RESULTS: The refractory period was 40 minutes for subject A, 70 minutes for subject B, and 95 minutes for subject C. The average refractory period across subjects was 68.33 minutes. The audible "crack" recovery was maintained for the remaining test days once the refractory period had been met. CONCLUSIONS: The audible "crack" heard during side posture lumbar manipulation is believed to originate from the zygapophyseal joints. This is supported by the presence of a refractory period and by the number of audible "cracks" found per manipulation.

Scavenging of reactive oxygen species by melatonin.

The direct effects of the neurohormone melatonin on reactive oxygen species (ROS) were investigated. Melatonin was found to inhibit DMPO-O-2 formation in a dose-dependent manner. At the level of 1. 7+/-0.07 mM, melatonin caused 50% inhibition of EPR signal intensity of DMPO-O-2 during the reaction of xanthine and xanthine oxidase. The reaction rate constant of melatonin with O2- was found to be 1.25+/-0.07x103 M-1 s-1. However, melatonin (up to 1.2 mM) did not exhibit significant effect toward OH radical, produced by the Fenton reaction. In addition, we found no evidence for the formation of the melatonin indolyl cation radical that presumably precedes conversion of melatonin to its stable N1-acetyl-N2-5-methoxykynuramine (AMK) metabolite following sequential reactions of melatonin with O2- and OH. On the other hand, melatonin was capable of scavenging H2O2 in a dose-dependent manner with an IC50=0.5+/-0.02 mM. The reaction rate constant of melatonin with H2O2 was found to be 2.52+/-0.19x105 M-1 s-1. Furthermore, melatonin was also found to inhibit 1O2-dependent 2,2,6,6-tetramethylpiperidine oxide (TEMPO) radical formation during rose bengal photodynamic reaction. The results suggest that melatonin's antioxidant properties, in part, may involve a direct effect on scavenging of ROS.

Association between CYP1A1 genotype, mRNA expression and enzymatic activity in humans.

Genetic susceptibility factors may play a role in determining adverse effects of exposure to environmental toxins. As a preliminary step to a molecular epidemiological study in a population exposed to 2,3,7,8-tetrachlorodibenzo-para-dioxin (TCDD), we investigated 20 healthy Caucasian volunteers with a set of putative susceptibility markers including a CYP1A1 Msp I restriction fragment length genetic polymorphism (RFLP), CYP1A1 mRNA expression, and ethoxyresorufin-O-deethylase (EROD) activity in cultured and mitogen-activated blood lymphocytes. Both basal (p = 0.008) and induced (p = 0.0001) EROD activity was significantly higher among persons with a mutation in one or both alleles of the CYP1A1 gene (variant CYP1A1 genotype). Induction in vitro by TCDD significantly increased EROD activity in both variant and wild-type CYP1A1 subjects; however, the absolute increase was greater in subjects with variant genotypes. An additive interaction between genotype and TCDD induction was suggested. Expression of CYP1A1 mRNA, both basal and induced, did not vary significantly across the genotypes.

Expression of the CYP1A1 gene in peripheral lymphocytes as a marker of exposure to creosote in railroad workers.

We have conducted a pilot study to assess levels of cytochrome CYP1A1 gene expression in human peripheral lymphocytes as a molecular biomarker assay for polycyclic hydrocarbon exposure. Basal and 3-methylcholanthrene-induced levels of gene expression were measured by standard slot-blot mRNA analyses in mitogen-stimulated cultures of peripheral blood lymphocytes from creosote-exposed railroad workers and unexposed control subjects. Dermal and inhalation exposure of workers to creosote may vary substantially as a function of working conditions related to temperature. Therefore, blood specimens were collected from separate groups during the winter, fall, and summer. Basal and induced CYP1A1 gene expression levels were not elevated in workers from any of the three seasonal studies. However, induced/basal (inducibility) CYP1A1 mRNA ratios from workers sampled in the summer (when actual exposures were greatest) were significantly higher when compared to those of controls (P < 0.01). These studies demonstrate the potential usefulness of specific gene expression assays in human peripheral lymphocytes for the assessment of carcinogen exposure in human populations.

Racial differences in restriction fragment length polymorphisms and messenger RNA inducibility of the human CYP1A1 gene.

Recent studies have examined the relationship between genetic polymorphisms of the human cytochrome P-4501A1 (CYP1A1) gene and lung cancer susceptibility. We have quantified genotypic frequencies and measured gene expression in the CYP1A1 gene within racially diverse groups in order to determine the relationship between genotype and transcriptional regulation of the CYP1A1 gene. Lymphocytes were obtained from 68 individuals of European-American, African-American, and Asian descent, and CYP1A1 gene inducibility was measured in mitogen-stimulated cells. CYP1A1 gene inducibility was significantly lower in African-Americans than in European-Americans or Asians, while several other population parameters were found to have no effect on gene expression levels. Restriction fragment length polymorphism analysis of lymphocyte DNA following MspI restriction enzyme digestion revealed a significant difference in the frequencies of CYP1A1 genotypes between European-Americans and Asians. The only homozygous variants detected were of Asian descent. The frequencies of CYP1A1 genotypes in all races conformed to Hardy-Weinberg genotypic equilibrium. When CYP1A1 gene inducibility was compared to CYP1A1 genotype, no significant correlations were found. These studies, along with our previous survey of CYP1A1 gene expression in creosote-exposed workers, add further support to the use of CYP1A1 gene inducibility as a potential marker of polycyclic aromatic hydrocarbon exposure in human populations.

CYP1A1 messenger RNA levels in placental tissue as a biomarker of environmental exposure.

The human CYP1A1 gene codes for an inducible enzyme system involved in biotransformation of certain xenobiotics, including polycyclic aromatic hydrocarbons; some of the metabolites are carcinogenic and mutagenic. Effects of environmental exposures (smoking, air pollution, and diet) on CYP1A1 gene induction in placental tissue and the modulation of induction by the CYP1A1 MspI RFLP were evaluated in two groups from Poland: 70 mother-child pairs from Krakow, a city with elevated air pollution; and 90 pairs from Limanowa, a less polluted area. Compared to placentas from nonsmoking women, CYP1A1 mRNA levels were significantly increased in placentas from current smokers (P < 0.001). Ex-smokers also had significantly higher placental mRNA levels, including women who quit smoking prior to pregnancy (P < 0.01). A marginal increase in CYP1A1 mRNA with environmental tobacco smoke exposure was evident. Within Krakow, there was an increase in CYP1A1 mRNA with ambient pollution at the place of residence for each woman, which was significant among women who were not employed away from the home (P < 0.05 controlling for smoking status, diet, and use of coal for heating). Significant increases in mRNA were associated with dietary consumption of smoked meat, cheese, and fish (P < 0.01). The CYP1A1 MspI RFLP was not a significant determinant of CYP1A1 mRNA levels after controlling for smoking and other variables. Human placenta provides a readily available and responsive system that can serve as a model for evaluating environmental and genetic determinants of CYP1A1 induction.

The detection of DNA-protein cross-links in rat tracheal implants exposed in vivo to benzo[a]pyrene and formaldehyde.

The DNA damage associated with benzo(a)pyrene (B[a]P) and formaldehyde (HCHO) exposure in rat tracheal implants was determined by alkaline filter elution adapted to measure DNA-protein cross-links (DPC) in vivo. In addition, histopathological responses of the tracheal epithelium were quantitated after multiple exposures to 20 micrograms B[a]P and 0.2% HCHO. Compared to either agent alone, combined exposure for 1-4 weeks caused an increase in cellular atypia and greater thickness of hyperplastic and metaplastic lesions. HCHO exposure resulted in a dose-dependent increase in DPC with a maximal response of 85% DNA filter retention at 0.2% HCHO, which were mostly removed by 72 h. B[a]P did not cause DPC, but when tracheas were pre-exposed to 20 micrograms B[a]P followed by 0.05% HCHO there was a 15% decrease in HCHO-induced DPC. This competition between B[a]P and HCHO for sites presumably on DNA does not offer a clear explanation for their markedly enhanced cocarcinogenicity observed in previous studies, but does demonstrate the interaction between the two agents in tracheal epithelium.

Occupational exposures to Cd, Ni, and Cr modulate titers of antioxidized DNA base autoantibodies.

This study was undertaken to establish whether occupational exposures to derivatives of carcinogenic metals evoke inflammatory immune responses, as determined by the presence of elevated titers of antibodies (Ab) that recognize oxidized DNA bases. Sera obtained from the blood of steel welders (Delaware) and from workers of the Centra Ni-Cd Battery Factory (Poznan, Poland) were analyzed by the enzyme-linked immunosorbent assay. To determine specific and nonspecific binding, an oxidized thymidine [5-hydroxymethyl-2'-deoxyuridine (HMdU)] coupled to bovine serum albumin (HMdU-BSA) as well as mock-coupled BSA (M-BSA) were used as antigens for coating the wells of microtiter plates. Titers of anti-HMdU Ab were significantly elevated in the high Cd and Ni exposure groups (18.3 +/- 3.2 vs 10.8 +/- 2.1 A492/microliters; p < 0.05). The sera of the groups with low exposures to Cd and Ni also had enhanced titers of those Ab but those increases were not statistically significant. Interestingly, the Ab titers present in the sera of controls for Cd and Ni exposures appear to be constant regardless of the protein content. In contrast, both lightly and heavily exposed subjects exhibited Ab titers that increased with increasing protein content. When 12 randomly selected workers (4 from each of the control, lightly, and heavily exposed groups) were outfitted with personal monitors, anti-HMdU Ab titers of those workers showed a significant difference between the groups with light (< 100 micrograms/m3) and heavy (> 200 micrograms/m3) exposures to Cd (9.8 +/- 3.7 vs 22.1 +/- 3.7 A492/microliters; p < 0.01) and Ni (11.7 +/- 1.4 vs 31.0 +/- 1.8; p < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Application of reliability models to studies of biomarker validation.

We present a model of biomarker validation developed in our laboratory, the results of the validation study, and the impact of the estimation of the variance components on the design of future molecular epidemiologic studies. Four different biomarkers of exposure are illustrated: DNA-protein cross-link (DNA-PC), DNA-amino acid cross link (DNA-AA), metallothionein gene expression (MT), and autoantibodies to oxidized DNA bases (DNAox). The general scheme for the validation experiments involves n subjects measured on k occasions, with j replicate samples analyzed on each occasion. Multiple subjects, occasions, and replicates provide information on intersubject, intrasubject, and analytical measurement variability, respectively. The analysis of variance showed a significant effect of batch variability for DNA-PC and MT gene expression, whereas DNAox showed a significant between-subject variability. Among the amino acids tested, cysteine and methionine showed a significant contribution of both batch and between-subject variability, threonine showed between-subject variability only, and tyrosine showed between-batch and between-subject variability. The total variance estimated through the experiment was used to calculate the minimum sample size required for a future epidemiologic study including the same biomarkers used for the reliability study. Such validation studies can detect the various components of variability of a biomarker and indicate needed improvements of the assay, along with possible use in field studies.

Relationship between ambient air pollution and DNA damage in Polish mothers and newborns.

Industrialized regions in Poland are characterized by high ambient pollution, including polycyclic aromatic hydrocarbons (PAHs) from coal burning for industry and home heating. In experimental bioassays, certain PAHs are transplacental carcinogens and developmental toxicants. Biologic markers can facilitate evaluation of effects of environmental PAHs on the developing infant. We measured the amount of PAHs bound to DNA (PAH-DNA adducts) in maternal and umbilical white blood cells. The cohort consisted of 70 mothers and newborns from Krakow, Poland, an industrialized city with elevated air pollution. Modulation of adduct levels by genotypes previously linked to risk of lung cancer, specifically glutathione S-transferase MI (GSTM1) and cytochrome P4501A1 (CYP1A1) Msp restriction fragment length polymorphism (RFLP), was also investigated. There was a dose-related increase in maternal and newborn adduct levels with ambient pollution at the women's place of residence among subjects who were not employed away from home (p < or = 0.05). Maternal smoking (active and passive) significantly increased maternal (p < or = 0.01) but not newborn adduct levels. Neither CYP1A1 Msp nor GSTM1 polymorphisms was associated with maternal adducts. However, adducts were significantly higher in newborns heterozygous or homozygous for the CYP1A1 Msp RFLP compared to newborns without the RFLP (p = 0.04). Results indicate that PAH-induced DNA damage in mothers and newborns is increased by ambient air pollution. In the fetus, this damage appears to be enhanced by the CYP1A1 Mspl polymorphism.

Role of H-ras in the malignant progression of rat tracheal epithelial cells.

The effect of an activated H-ras oncogene on the progression of neoplasia was studied in transformed rat tracheal epithelial cells. Nude mouse tumours produced by injection of these cells exhibited a higher fraction of cells containing the mutant ras gene that did the injected cells, while a subclone that lacked the ras mutation was much less tumorigenic than parental cells. Serial passage of one cell line containing a ras mutation resulted in an increase in the fraction of ras-mutated cells, which suggests that, in this line, ras activation may confer a selective advantage in vitro as well. However, this was not seen in another ras-containing line, suggesting the importance of alternative pathways in malignant progression of rat tracheal epithelial cells.

Benzo[a]pyrene- and formaldehyde-induced DNA damage and repair in rat tracheal epithelial cells.

The single and combined effects of 2 environmental carcinogens, benzo[a]pyrene (BAP) and formaldehyde (HCHO), on cell growth and DNA damage were assessed in a rat tracheal epithelial cell line, C18. Treatment of C18 cells with HCHO for 90 min reduced the calculated growth index at the highest concentration tested, 400 microM, while no growth effects were observed with BAP treatments. Combination treatments reduced the growth index to 75% of control values. Alkaline elution analysis of C18 cell DNA detected both DNA-protein crosslinks (DPC) and single-strand breaks (SSB) as a result of HCHO treatment, while BAP caused only SSB. HCHO-induced SSB were repaired within 2 h, whereas BAP-induced SSB persisted for at least 48 h. Combination treatment of cells with BAP followed by HCHO enhanced the number of SSB, but reduced DPC. The results are discussed in reference to earlier work which demonstrated the interaction in vivo between BAP and HCHO with respect to their carcinogenicity in rat tracheal implants.

A quantitative metabolic inhibition test for screening toxic compounds with cultured cells.

The toxicity of substances for monolayer cultures of rat lung fibroblasts with phenol red as a pH indicator in the medium was measured by comparing the changes produced in medium color against 10 stable color standards. The toxicities of 6 concentrations each of actinomycin D, amphotericin B, chloroquine, 2-deoxyglucose, oligomycin, puromycin, and silicon dioxide were measured at 12 h and daily for 7 days on the same cultures throughout. Morphologic changes were monitored at the same times. The method, which simultaneously measures both concentration and time effects, was rapid, simple-to-perform, reproducible, low-in-cost, and quite sensitive. Its reproducibility depended on details of the cell culture methodology. The method is unsuitable for acidic or basic substances, or substances poorly-soluble in culture medium. Unless combined with morphologic evaluation, substances producing delayed toxicity may give misleading results.

A model system for measuring comparative toxicities of cardiotoxic drugs with cultured rat heart myocytes, endothelial cells and fibroblasts. I. Emetine, chloroquine and metronidazole.

Neonatal rat hearts were separated into separate cultures of beating myocytes (M cells), endothelial cells (E cells) and fibroblasts (F cells). Their susceptibilities to the toxic effects of emetine, chloroquine and metronidazole were then compared using a quantitative metabolic inhibition test (QMIT) and morphologic and beating changes as indices of injury. Measurements on the same cultures were made at 6 h and 12 h daily for 7 days with E and F cells; with M cells for 3 days. Metronidazole was non-toxic for all cell types at 810 micrograms/ml, whether as the parent compound or after attempted rat liver microsomal activation. QMIT data, integrated as time-concentration effects, showed all cell responses to either emetine or chloroquine to be parallel (P less than 0.05), and their order of susceptibility to be: E greater than M greater than F cells. Although morphologic signs of injury and changes in beating are not readily evaluated statistically, there was a general parallelism between these indices of injury and those of QMIT. As judged by QMIT emetine was more toxic than chloroquine. However, at equivalent QMIT grades chloroquine produced greater effects on morphology and beating. Toxic concentration-50 values for chloroquine with the QMIT were similar to reported human toxic and lethal blood concentrations.

A model system for measuring comparative toxicities of cardiotoxic drugs for cultured rat heart myocytes, endothelial cells and fibroblasts. II. Doxorubicin, 5-fluorouracil and cyclophosphamide.

Separate cultures of beating myocytes (M cells), endothelial cells (E cells) and fibroblasts (F cells) from neonatal rat hearts were compared for their susceptibilities to the toxic effects of doxorubicin, 5-fluorouracil and cyclophosphamide. Toxic injury was measured as inhibition of metabolism and changes in morphology for all cell types, and changes in beating of myocytes. Cells were exposed to the anti-tumor agents for 3 days with myocytes and 7 days for endothelial cells and fibroblasts. All measures of injury were made several times the first day and daily thereafter, with the same cultures used throughout. Toxic effects of DOX and 5-FU were greater for E and F cells than for M cells, and all measures of toxicity were generally parallel. Cyclophosphamide, whether activated by liver microsomes or unactivated, was less toxic in general than the other agents, but it was more toxic for M cells than for E cells or F cells.

An experimental model for oncogene activation during tumor progression in vivo.

Tumor progression usually involves a complex pattern of molecular alterations. In many human tumors oncogene amplification or activation has been associated with advanced stages of cancer. Transfection studies have demonstrated the ability of several cellular oncogenes to induce a more malignant phenotype in transformed cells. We have examined the role of c-myc in tumor progression in rat tracheal cell culture, and in rat skin tumors induced by ionizing radiation. In the latter in vivo model, c-myc amplification was found to occur as a function of tumor size. Serial biopsies of growing tumors confirmed the trend toward increased gene copy number with time and stage of progression. This effect was specific for the c-myc gene, in epithelial tumors. Evidence was found for a role of tumor heterogeneity and evolution of tumor cell subpopulations in determining the oncogene activation profile of individual tumors.

Growth inhibition and DNA damage induced by benzo[a]pyrene and formaldehyde in primary cultures of rat tracheal epithelial cells.

The effects of benzo[a]pyrene (BAP) and formaldehyde (HCHO), alone and combined, on cell growth and DNA damage were determined in primary cultures of rat tracheal epithelial cells dissociated from rat tracheas. Cell cultures treated with 25 microM BAP for 24 h or 200 microM HCHO for 90 min did not have a marked reduction in cell growth. However, their combined treatment reduced cell growth by 60% of control when cultures were exposed to BAP followed by HCHO as well as the reverse order. None of these treatments significantly decreased cell viability as judged by dye exclusion, nor did they enhance cell terminal differentiation as measured by cornified envelope formation. Alkaline elution analysis of DNA damage detected both DNA-protein crosslinks (DPC) and DNA single-strand breaks (SSB) as a result of HCHO treatment, whereas BAP treatment caused only SSB. While HCHO-induced SSB were repaired within 2 h, BAP-induced SSB were detected 3 days after treatment. Combined treatment of cell cultures with BAP followed by HCHO resulted in more SSB than was obtained from either agent alone, but less DPC than was detected from HCHO alone. The increased number of SSB obtained from this combined treatment may be related to the marked enhancement of carcinogenesis observed in earlier in vivo-in vitro studies.

The detection of DNA-protein complexes in vitro by an immunological assay.

We have developed an immunological assay to detect DNA-protein complexes (DPCs) in cell cultures treated with environmental toxicants. The assay uses an antiserum developed against K(2)CrO(4)-induced DPCs, which recognizes an acidic protein with a molecular weight of 95 kDaltons. The method uses a filter-binding assay to trap the DPCs from SDS-lysed cell cultures on polycarbonate filters, after which they are immunologically probed with the DPC antiserum. Cultures of Chinese hamster ovary cells were treated with K(2)CrO(4), formaldehyde or UV irradiation. DPCs were detected immunologically in cells treated with K(2)CrO(4) or UV irradiation, but not in those treated with formaldehyde. These results were similar to those obtained when DPCs were detected by filter-binding assay using radiolabelled cell cultures to measure the adherence of protein and DNA to the filters. In addition, both methods gave analogous dose responses of DPC formation in K(2)CrO(4)-treated cells. This novel immunological assay for detecting DNA lesions allows the rapid analysis of samples, which do not need to be radiolabelled, and thus it may have an application as a non-invasive test to screen for DNA-protein complexes.

Induction of metallothionein and heme oxygenase in rats after inhalation of endotoxin.

Various stress proteins appear to play a role in injury and repair produced by inhaled pollutants. The present study examined the effect of inhaled endotoxin on the expression of the metallothionein and heme oxygenase genes. Rats were exposed to saline or endotoxin aerosols for 3 h and sacrificed up to 3 d following exposure. The significant induction of metallothionein mRNA in both the lung (fourfold increase) and liver (one-fold) were greatest at 3 h and returned to basal levels by 24 h after endotoxin exposure. Similarly, the increase in tissue metallothionein was greater in the lung. In situ hybridization in mice showed large increases in the relative abundance of metallothionein transcripts in epithelial cells of the conducting airways, in surrounding airway tissue, and in the nearby gas exchange region. While an endotoxin-induced significant increase in heme oxygenase mRNA followed a time course similar to that observed for metallo thionein, the relative magnitude was reversed for the lung and liver. Heme oxygenase mRNA was induced greater in the liver (twofold) than in the lung (60% above control). Our findings demonstrate that metallothionein and heme oxygenase are early response genes that are rapidly activated after inhalation of occupationally relevant concentrations of endotoxin.

The induction of growth-altered cell populations (tumor-initiation sites) in rat tracheal implants exposed to benzo[a]pyrene and formaldehyde.

The effects of exposure to benzo[a]pyrene (BAP) and formaldehyde (HCHO), alone and combined, on the induction of carcinogenesis in rat tracheal implants was determined as the number of growth-altered cell populations (tumor-initiation sites, TIS) per trachea. While exposure twice-weekly for 4.5 months to 0.2% HCHO solution gave only a weak response (0.25 TIS/trachea), 2.37 TIS per trachea were detected after exposure to 20 micrograms BAP in the same regimen. The combination of BAP followed by HCHO had a greater response than either agent alone (7.83 TIS/trachea), while the reverse exposure gave 1.49 TIS per trachea, which was less than BAP alone. Thus, the induction of TIS by combined exposure to BAP and HCHO was dependent on the order of exposure, and could not be predicted from their individual exposures.