Comparison of internal dose measures of solvents in breath, blood and urine and genotoxic changes in aircraft maintenance personnel.

Solvents and fuels are in widespread use both in civilian and military populations. 1,1,1-trichloroethane (TCA), xylene, toluene, methyl ethyl ketone (MEK) and methylene chloride are found in a variety of compounds including degreasing agents, paints, coatings, pesticides and paint strippers. Toluene and xylene are also found in fuels, which are complex mixtures of hundreds of agents. The purpose of this investigation was twofold. The first was to determine the optimum medium to measure internal dose of solvents comparing blood, urine and breath. The second was to determine if low level exposures were associated with genotoxic changes after a short-term exposure of fifteen or thirty weeks. To accomplish the first goal a pilot study was initiated involving eight volunteers who worked in aircraft maintenance including sheet metal, painting and assembly mechanic jobs. Industrial hygiene measurements were evaluated over 30 working days. Breath, blood and a 24-hour urine sample were collected twice to compare internal dose parameters. To achieve the second goal, 58 newly hired subjects were monitored prior to exposure and over 30 weeks to determine if there were genotoxic changes as a result of solvent and/or fuel exposure as measured by sister chromatid exchanges (SCEs) and micronuclei (MN). Exposure groups included workers involved in sheet metal (fuel cell) activities, painting, fueling operations and flight line. Results of the pilot study demonstrated that industrial hygiene air samples and internal breath measures taken on the same day were highly correlated for measuring TCA (r = 0.93) and toluene (r = 0.90) but was not as well correlated for the other compounds. Breath measures were more sensitive for measuring low level exposure than were either analytes in blood or 24-hour urine samples; these latter two measures were usually below the limit of detection. A small but statistically significant increase in the frequency of SCEs occurred after 30 weeks of exposure for sheet metal workers (p = 0.003) and for painters (p = 0.05). The MN frequency in the sheet metal workers initially showed a significant increase by 15 weeks, but by 30 weeks had decreased. Chance occurrence of exposures to other occupational or non-occupational agents can not be eliminated as a cause of the genotoxic results since between 58 and 93 total analytes could be found in the breath of some aircraft maintenance personnel.

Titanium osseointegrated implants combined with hyperbaric oxygen therapy in previously irradiated mandibles.

STATEMENT OF PROBLEM: Treatment for head and neck malignancies commonly involves radiation therapy. As a result of this therapy the vascular supply to irradiated structures is altered and results in decreased tissue perfusion. In addition to vascular changes, bony structures undergo a reduction in osteoblastic and osteoclastic activity. These tissue alterations, especially in the mandible, enhance the risk of osteoradionecrosis. To avoid this occurrence, many patients who have undergone radiation therapy do not receive elective preprosthetic surgeries, including implant therapy. PURPOSE OF STUDY AND METHODS: This report presents the preliminary results of placing 18 titanium screw implants into previously irradiated mandibles in conjunction with hyperbaric oxygen therapy. RESULTS: Of the 18 implants placed, 17 (94%) were judged to be osseointegrated at the abutment connection. One implant did not receive an abutment and was "put to sleep." The remaining 16 (88%) were used for prosthetic rehabilitation. CONCLUSION: The use of implants in irradiated tissues may provide a means of enhancing prosthetic rehabilitation while reducing the risk of tissue trauma that may develop into osteoradionecrosis.

Molecular modeling parameters predict antioxidant efficacy of 3-indolyl compounds.

Many dietary constituents, such as indole-3-carbinol, are chemoprotective in toxicity and carcinogenicity bioassays. Indole-3-carbinol and related congeners appear to protect partly via radical and electrophile scavenging. To develop better chemoprotective indoles with lower intrinsic toxicity, we performed molecular graphic and quantum-mechanical analyses of model indolyl compounds to ascertain the determinant molecular features for antioxidant activity. We examined eight structurally related 3-indolyl compounds for relationships between antioxidation potential (using in vitro lipid peroxidation assays) and electronic, polar, and steric parameters, including bond dissociation energies, bond lengths, dipole moments, electronic charge densities, and molecular size parameters. Electronic features of the 3-methylene carbon and 1-nitrogen were not predictive of antioxidative potency due to extensive charge delocalization of the cation radical following electron abstraction from the nitrogen. Antioxidant efficacy of 3-indolyl compounds was most strongly predicted by molecular size parameters and by the energy of electron abstraction as calculated from the difference in heat of formation between the parent compound and its cation radical. A highly predictive multiple linear regression correlation model (r2 = 0.97) was obtained using the parameters of heat of formation, molecular weight, log P, and diplole moment.

Interaction between the Ah receptor and proteins binding to the AP-1-like electrophile response element (EpRE) during murine phase II [Ah] battery gene expression.

We have studied three Phase II genes in the mouse dioxin-inducible [Ah] battery: Nmo1 [encoding NAD(P)H:menadione oxidoreductase], Ahd4 (encoding the cytosolic aldehyde dehydrogenase ALDH3c), and Ugt1*06 (a UDP glucuronosyltransferase). Oxidant-induced Nmo1 gene expression in the c14CoS/c14CoS mouse appears likely to be caused by homozygous loss of the fumarylacetoacetate hydrolase (Fah) gene on Chr 7 and absence of the enzyme (FAH), which leads to increased levels of endogenous tyrosine oxidative metabolites. We show here that increases in [Ah] Phase II gene expression in the 14CoS/14CoS mouse are correlated with an AP-1-like DNA motif called the electrophile response element (EpRE), which has been found in the 5' flanking regulatory regions of all murine (Ah) Phase II genes. Aromatic hydrocarbon response element (AhREs) are responsible for dioxin-mediated upregulation of all six [Ah] battery genes, and one or more AhREs have been found in the 5' flanking regulatory regions of all of these [Ah] genes. Gel mobility shift assays, with a synthetic oligonucleotide probe corresponding to the EpRE, show that EpRE-binding proteins are more than twice as abundant in 14CoS/14CoS than in the wild-type ch/ch nuclear extracts. Competition studies of EpRE-specific binding with an excess of EpRE, mutated EpRE, AP-1, AhRE3, mutated AhRE3, and C/EBP alpha oligonucleotides suggest that several common transcriptional factors bind to the EpRE and AhRE3 motifs. Two monospecific antibodies to the Ah receptor (AHR) protein block formation of an EpRE-specific complex on gel mobility electrophoresis. These data suggest that AHR (or AHR-related protein) might be an integral part of the EpRE-binding transcriptional complex associated with the oxidative stress response. To our knowledge, this is among the first reports of the same transcription factor operating at two different response elements upstream of a single gene.

Enzyme induction by L-buthionine (S,R)-sulfoximine in cultured mouse hepatoma cells.

Induction of Phase II enzymes of the [Ah] gene battery by L-buthionine (S,R)-sulfoximine (BSO) and other agents was examined in mouse hepatoma Hepa-1c1c7 cells. BSO, a nonelectrophilic inhibitor of gamma-glutamylcysteine synthetase (GCS), is routinely used to examine the toxicological implications of GSH depletion. Exposure to BSO for 24 h produced a 75-85% depletion of GSH levels, proportional to the inhibition of GCS activity, as well as small increases in the UDP-glucuronosyltransferase (UGT, 60%) and glutathione transferase (GST, 30%) enzyme activities in Hepa-1 wild-type (wt) cells. However, for the NAD(P)H:menadione oxidoreductase (NMO1) and cytosolic aldehyde dehydrogenase class 3 (AHD4) enzyme activities, BSO produced larger increases (110% and 170%, respectively). The mechanisms of NMO1 and AHD4 induction were examined further. In Hepa-1 wt cells, NMO1 and AHD4 activities were increased by the aromatic hydrocarbon inducer 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and by the electrophile tert-butylhydroquinone (tBHQ), known inducing agents for these enzymes. However, NMO1 and AHD4 were induced in Ah receptor nuclear translocation-defective mutant (c4) cells by BSO and tBHQ, but not by TCDD, suggesting that the induction by BSO and tBHQ is not Ah receptor-mediated. In wt cells, N-acetylcysteine produced a concentration-dependent increase in intracellular cysteine levels, but not GSH levels, in the absence or presence of BSO. Furthermore, N-acetylcysteine had no effect on NMO1 activity under any conditions examined, suggesting that GSH levels per se, rather than change in overall thiol status, might be mediating increased NMO1 activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of [Ah] gene battery enzymes and glutathione levels by 5,10-dihydroindeno[1,2-b]indole in mouse hepatoma cell lines.

The murine aromatic hydrocarbon ([Ah]) gene battery consists of at least six genes that code for two functionalizing (Phase I) enzymes and four non-functionalizing (Phase II) enzymes. These enzymes are induced by compounds such as aromatic hydrocarbons and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) that bind to the cytosolic Ah receptor protein. Studies in rodents indicate that certain enzymes of this battery, namely cytochrome P4501A1 (CYP1A1), UDP-glucuronosyltransferase (UGT1*06) and NAD(P)H: quinone acceptor oxidoreductase (NMO1) are induced by the synthetic antioxidant 5,10-dihydroindeno[1,2-b]indole (DHII). The induction of [Ah] gene battery enzymes and the levels of reduced glutathione (GSH) were examined in mouse Hepa-1c1c7 hepatoma wild-type cells (wt), a CYP1A1 metabolism-deficient mutant (c37) and an Ah receptor nuclear translocation-defective mutant (c4). DHII and TCDD increased the activities of ethoxyresorufin O-deethylase, an indicator of CYP1A1 activity, as well as NMO1, UGT1*06, cytosolic aldehyde dehydrogenase class 3 and glutathione S-transferase form A1 in wt cells, but had little or no induction effect in c37 or c4 cells. DHII and TCDD differed in their effects on GSH levels; while DHII increased GSH levels 3-fold in wt, but not at all in c37 or c4 cells, TCDD had no effect on GSH levels in any cell type. However, GSH levels were enhanced in both wt and c4 cells by tert-butyl hydroquinone (TBHQ). L-Buthionine S,R-sulfoximine, an inhibitor of gamma-glutamylcysteine synthetase, prevented DHII-induced increases in wt cell GSH. The increase in GSH levels occurred after 8 h, while the induction of enzymes occurred within 4 h. The induction of the higher GSH levels in wt cells by DHII and TBHQ correlated with increases in intracellular levels of the GSH precursor thiol cysteine, as well as with increased activities of gamma-glutamylcysteine synthetase, the rate-limiting enzyme of GSH synthesis. However, TBHQ-mediated GSH increases in c4 cells were accompanied by increased gamma-glutamylcysteine synthetase activity with no change in intracellular cysteine concentration. The results suggest that DHII induction of [Ah] gene battery enzymes requires a functional Ah receptor, but not the functional gene product CYP1A1. Furthermore, metabolism, possibly via CYP1A1, appears to be required for DHII to enhance intracellular levels of cysteine and GCS activity that result in higher GSH levels.

Mechanisms of protection from menadione toxicity by 5,10-dihydroindeno[1,2,-b]indole in a sensitive and resistant mouse hepatocyte line.

Established cell lines derived from newborn livers of c14CoS/c14CoS and cch/cch mice have been shown to be genetically resistant (14CoS/14CoS cells) or susceptible (ch/ch cells) to menadione toxicity. These differences are due in part to relatively higher levels of reduced glutathione (GSH) and NAD(P)H:menadione oxidoreductase (NMO1) activity in the 14CoS/14CoS cells. The indolic membrane-stabilizing antioxidant 5,10-dihydroindeno[1,2-b]indole (DHII) was shown previously to protect against various hepatotoxicants in vivo and in primary rat hepatocytes. This report describes how the 14CoS/14CoS and ch/ch cell lines provide a valuable experimental system to distinguish the mechanism of chemoprotection by DHII from menadione toxicity. The addition of 25 microM DHII produced a time-dependent decrease in menadione-mediated cell death in 14CoS/14CoS cells, with little effect on ch/ch cell viability. The maximum protective effect occurred at 24 hr, although the concentration of DHII remained constant for 48 hr. The protective effect of DHII correlated with enhanced glutathione levels (234% increase at 24hr), as well as induction of four enzymes involved in the detoxification and excretion of menadione: NAD(P)H:menadione oxidoreductase (NMO1, quinone reductase), glutathione reductase, glutathione transferase (GST1A1), and UDP glucuronosyltransferase (UGT1*06), with 24-hr maximum induction of 707, 201, 171 and 198%, respectively. Other biotransformation enzymes not directly involved in menadione metabolism (glutathione peroxidase, cytochromes P4501A1 and P4501A2, copper-, zinc-dependent superoxide dismutase, and NADPH cytochrome c oxidoreductase) were not induced by DHII. Menadione-stimulated superoxide production was inhibited 50% by DHII only in 14CoS/14CoS cells, and the inhibition required 24-hr preincubation. Pretreatment with DHII also protected both cell types against the menadione-mediated depletion of GSH, and the increase in percent (oxidized glutathione GSSG), an indicator of oxidative stress. These results suggest that DHII does not protect against menadione toxicity by virtue of its antioxidant or membrane-stabilizing properties. Rather, it acts by inducing a protective enzyme profile that migates redox cycling and facilitates excretion of menadione.

Odontogenic myxoma of the maxillary sinus: a clinical report.

This report describes the transoral maxillary resection of an odontogenic tumor and prosthetic reconstruction with titanium implants placed horizontally in the residual palate. The treatment provided enabled the removal of the lesion and prosthetic restoration of the missing structures, while maintaining optimal facial esthetics and obtaining support, retention, and stability for the prosthesis (Fig. 5).

Exogenous iron overload in perinatal hemochromatosis: a case report.

We report an infant with fatal neonatal liver disease in whom efforts to correct an associated unremitting anemia resulted in massive exogenous iron overload that was expressed as perinatal hemochromatosis (PH). Levels of iron and copper were elevated in multiple tissues. Echovirus subtype 9, recovered from the urine at age 3 weeks, may have been etiologic in the liver failure. PH is best viewed as a definable phenotype with an undefined genetic and/or environmental basis that emerges only in the context of severe perinatal liver disease. The absence of hemosiderin in splenic and bone marrow reticuloendothelial (RE) cells of our patient suggests an important role for RE cell dysfunction.

Studies of the reactivity of morpholine, 2-mercaptobenzothiazole and 2 of their derivatives with selected amino acids.

Previous studies in guinea pigs showed that DTDM, MMBT and MBT were sensitizing agents. The purpose of this study was to investigate the biochemical basis of this sensitization reaction. The reactions of representative amino acids, Cys, Lys & Gly, with morpholine, DTDM, MMBT & MBT were examined. New compounds were found between MMBT and all 3 amino acids. Additional new compounds were seen for Cys/MBT, Cys/DTDM, Lys/MBT & Gly/DTDM. No reaction was observed with morpholine. The new compounds were examined spectroscopically. Results suggest that the sensitizing nature of DTDM is probably a reaction of its disulfide bond with the -SH & -NH2 groups of amino acid moieties. The conjugation of Cys/MMBT may be via the same reaction mechanism. The -SH group of MBT may react either via oxidation and/or thioester formation with the carboxylate group of amino acids. These results also suggest that in vitro tests may predict the sensitizing potential of certain compounds.

Nucleophilic index value: implication in the protection by indole-3-carbinol from N-nitrosodimethylamine cyto and genotoxicity in mouse liver.

A novel assay system was developed in order to quantitate the nucleophilicity of pure chemicals or tissue extracts. This Nucleophilic Index Value (NIV) assay was based on the ability of putative nucleophiles to inhibit the methylation of cysteine by limiting concentrations of the electrophilic source, N-methyl-N-nitrosourea (MNU). Efficacy of model and cellular nucleophiles was quantitated as nmol cysteine protected by the nucleophile from methylation by MNU/h/mM compound. The NIVs of the pure compounds ascorbate, glutathione, 4-(4-nitrobenzyl)-pyridine (NBP) and indole-3-carbinol (I-3-C) were 2400, 1600, 3 and 0, respectively. When mice were treated with I-3-C by gavage at dosages of 0, 25, 50, 75 or 100 mg/kg body wt, the NIV for ethyl acetate extracts of the livers 1 h after treatment were 0, 33, 47, 52 and 92 nmol cysteine preserved/h/g tissue, respectively. The I-3-C enhancement of NIV was not attributable to ascorbate or glutathione, neither of which were present in the ethyl extracts of liver. When mice were treated with 10 mg N-nitrosodimethylamine (NDMA)/kg body wt 1 h after the varying dosages of I-3-C, the 24 h post-NDMA plasma alanine transaminase (ALT) values were decreased by I-3-C pretreatment in a dose-dependent fashion. Plasma ALT values were used in this study as an indicator of hepatotoxicity. The coefficient of determination, r2, computed from the linear least squares correlation coefficient between NIV and ALT values, was 0.80 (0-100 mg I-3-C/kg) and 0.97 (0-75 mg/kg).(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of diet on the expression of hepatotoxicity from carbon tetrachloride in ICR mice.

Carbon tetrachloride-mediated hepatotoxicity in mice was influenced by two standard, commercially available diets and by a corn oil treatment vehicle. Animals maintained on Purina 5001 diet were less sensitive than animals maintained on Teklad LM-485 diet to hepatic intoxication by carbon tetrachloride (CCl4). Lower sensitivity of the Purina group was evidenced by significantly lower plasma alanine aminotransferase (ALT) levels and higher hepatic cytochrome P-450 levels at all dosages of CCl4. In addition to the diets, the nature of the corn oil vehicle affected toxicological responses of mice to CCl4. When the vehicle from which tocopherols had been extracted was used, CCl4 elicited about twice the levels of plasma ALT than when nonextracted corn oil was used. In conclusion, the nature of the animal diet and treatment vehicle not only can influence toxicological response, but also can be important considerations in the interpretation of toxicological data.

Intervention in free radical mediated hepatotoxicity and lipid peroxidation by indole-3-carbinol.

The cytoprotective effect of the natural dietary constituent indole-3-carbinol (I-3-C) on carbon tetrachloride (CCl4) mediated hepatotoxicity in mice was examined. I-3-C pretreatment by gavage 1 hr prior to intraperitoneal injection of CCl4 produced a 63% decrease in CCl4-mediated centrolobular necrosis and a related 60% decrease in plasma alanine aminotransferase activity (a marker of liver necrosis). Since the toxicological effects of CCl4 are mediated by radical species generated during reductive metabolism by cytochrome P-450, we examined the potential ability of I-3-C to scavenge reactive radicals. Three systems were used to evaluate the ability of I-3-C to intervene in free radical mediated lipid peroxidation. These systems consisted of the following: (1) phospholipid dissolved in chlorobenzene, with peroxidation initiated by the thermal and photo decomposition of azobisisobutyronitrile (AIBN); (2) sonicated phospholipid vesicles in phosphate buffer (pH 7.4), with peroxidation initiated by ferrous/ascorbate; and (3) mouse liver microsomes containing an NADPH-regenerating system, with peroxidation initiated with CCl4. Lipid peroxidation was measured in these three systems as thiobarbiturate-reacting material. In the AIBN and ferrous/ascorbate systems, I-3-C inhibited lipid peroxidation, with greater inhibition under conditions of low rates of free radical generation. I-3-C was not as effective an antioxidant as butylated hydroxytoluene (BHT) or tocopherol, but it inhibited peroxidation in a dose-response manner. I-3-C was most effective as a radical scavenger in the microsomal CCl4-initiated system by inhibiting lipid peroxidation in a dose-dependent fashion, with 50% inhibition at 35-40 microM I-3-C. This concentration is about one-third of the concentration of I-3-C achieved in liver after treatment of mice by gavage with 50 mg I-3-C/kg body weight. These data suggest that I-3-C may be a natural antioxidant in the human diet and, as such, may intervene in toxicological or carcinogenic processes that are mediated by radical mechanisms.

Protection from N-nitrosodimethylamine-mediated liver damage by indole-3-carbinol.

Indole-3-carbinol (I-3-C) was examined for its ability to protect mice against 24-hr N-nitrosodimethylamine (NDMA)-mediated hepatotoxicity. NDMA (20 mg/kg body weight) alone produced extensive hemorrhagic and centrolobular necrotic lesions, with a necrotic severity index of 3.0 +/- 0.4 (scale of 0-5). Treatment with 50 mg/kg body weight of I-3-C by gavage, 1 hr prior to NDMA, substantially protected against hemorrhagic lesions. Furthermore, I-3-C lowered the NDMA-mediated tissue necrotic index to 1.5 +/- 0.3, by reducing the extent of tissue necrosis rather than the severity in the necrotic region. Release of liver enzymes into the blood correlated with the histopathology; I-3-C reduced NDMA-mediated elevated activities of plasma alanine transaminase and ornithine transcarbamylase by 84 and 51.3%, respectively. Although no changes in nonprotein sulfhydryls were evident at 24-hr after NDMA, ascorbate levels were reduced to 40% of control values. However, treatment with I-3-C prior to NDMA prevented the decline in tissue ascorbate concentrations. In vitro, I-3-C was found to be a type II ligand for cytochrome P-450, with a Ks value of 237 microM. However, if such binding occurs in vivo, it does not protect against the approximately 60% decrease in hepatic cytochrome P-450 or the 80% decrease in NDMA demethylase I activity produced by NDMA. Since I-3-C slightly enhances cytochrome P-450 content and NDMA demethylase activity, the histopathologic protection by I-3-C must be due to factors other than inhibiting metabolic activation of NDMA.

Protection against carbon tetrachloride hepatotoxicity by pretreatment with indole-3-carbinol.

The effects of administering indole-3-carbinol (I-3-C) on carbon tetrachloride (CCl4)-induced hepatotoxicity were examined. Mice received by gavage 0-150 mg I-3-C/kg body wt in methanol-extracted corn oil, followed 1 h later by 15 microliters CCl4/kg body wt in corn oil. Animals were sacrificed 24 h after receiving CCl4. Pretreatment with I-3-C reduced the degree of centrolobular necrosis, as observed histologically. Additionally, CCl4-mediated elevated serum enzymes were reduced by I-3-C. Although I-3-C induced elevated levels of cytochrome P-450 and associated mixed-function oxidase activity, the CCl4 depression of these parameters was not clearly reversed by I-3-C. However, CCl4 produced decreases in hepatic levels of glutathione (GSH), total reducing equivalents, and protein sulfhydryls, all of which were restored to control levels by I-3-C. Using mouse liver microsomes in an NADPH-fortified reaction mixture, I-3-C inhibited, in a concentration-dependent manner, CCl4-initiated lipid peroxidation, with 50% inhibition at 35-40 microM I-3-C. When mice were treated by gavage with 50 mg [14C]I-3-C/kg body wt, concentrations of radiolabel in the liver were greater than 100 microM after 1 hr. This was five times the level of radioactivity measured in blood and three times the concentration of I-3-C necessary for 50% inhibition of CCl4-mediated lipid peroxidation in vitro. The data are consistent with the hypothesis that I-3-C intervenes in CCl4-mediated hepatic necrosis by combining with reactive free radical metabolites of CCl4, thereby protecting critical cellular target sites.

Indole-3-carbinol inhibits lipid peroxidation in cell-free systems.

Free radicals mediate toxicological and carcinogenic responses of tissues to many chemicals. Cellular defenses against radical mediated damage utilize endogenous substances such as tocopherol, ascorbate and GSH. Here we report a new antioxidant, indole-3-carbinol (I-3-C), a natural constituent of human diet. In chlorobenzene containing soy phospholipids, lipid oxidation was initiated with azobisisobutyronitrile; I-3-C inhibited formation of thiobarbituric acid-reactive material in a dose-dependent manner. Similar results were obtained in an aqueous system containing phospholipid vesicles initiated by Fe/ascorbate. For both systems I-3-C was less effective than tocopherol or BHT as antioxidant. To assess these antioxidant effects in vivo, mice were treated with I-3-C by gavage. A hepatic post-mitochondrial supernatant fraction isolated 2 hours after treatment showed dose-dependent decreases in NADPH-mediated lipid oxidation which correlated with decreases in 14C-nitrosodimethylamine covalent binding to protein. Although hepatotoxicity may not involve lipid oxidation per se, it does indicate that free radical damage had occurred. Inhibition of damage by I-3-C suggests that this dietary component has the potential to ameliorate radical mediated chemical toxicity.