Maxillary sinus floor elevation with bovine bone mineral combined with either autogenous bone or autogenous stem cells: a prospective randomized clinical trial.

AIM: To assess whether differences occur in bone formation after maxillary sinus floor elevation surgery with bovine bone mineral (BioOss(®)) mixed with autogenous bone or autogenous stem cells. The primary endpoint was the percentage of new bone three months after the elevation procedure. MATERIAL AND METHODS: In a randomized, controlled split-mouth design, in 12 consecutive patients (age 60.8 ± 5.9 years, range 48-69 years) needing reconstruction of their atrophic maxilla, a bilateral sinus floor augmentation procedure was performed. Randomly, on one side the augmentation procedure was performed with bovine bone mineral (BioOss(®)) seeded with mononuclear stem cells harvested from the posterior iliac crest (test group) while BioOss(®) mixed with autogenous bone (harvested from the retromolar area) was applied on the contra-lateral side (control group). On 14.8 ± 0.7 weeks after the sinus floor elevation, biopsies from the reconstructed areas were taken at the spots where subsequently the endosseous implants were placed. The biopsies were histomorphometrically analyzed. RESULTS: Significantly more bone formation was observed in the test group (17.7 ± 7.3%) when compared with the control group (12.0%± 6.6; P=0.026). In both the test and control group, all implants could be placed with primary stability. In one patient, not all biopsies contained BioOss(®). This patient was excluded from analysis. CONCLUSION: Mesenchymal stem cells seeded on BioOss(®) particles can induce the formation of a sufficient volume of new bone to enable the reliable placement of implants within a time frame comparable with that of applying either solely autogenous bone or a mixture of autogenous bone and BioOss(®). This technique could be an alternative to using autografts.

River quality assessment implications of a prototype project.

The U.S. Geological Survey recently completed an intensive river quality assessment study of the Willamette River basin, Oregon. The most noteworthy finding was that across-the-board advanced waste treatment was not the answer to the problem of meeting stringent water quality standards established for the Willamette River. This implies that rigid nationwide standards and regulations are likely to result in unneeded expenditures in some river basins and in unachieved standards in others. It was also found that existing water quality data collected under monitoring- and surveillance-type programs are inadequate for defining the critical cause-effect relationships that control river quality problems. Intensive, synoptic surveys keyed to local problems and conditions are required to provide an adequate information base for making key management decisions.

[Polymeric implant materials for the reconstruction of tracheal and pharyngeal mucosal defects in head and neck surgery]. / Polymere Implantatmaterialen zur trachealen und pharyngealen Schleimhautrekonstruktion in der Kopf-Hals-Chirurgie.

The existing therapeutical options for the tracheal and pharyngeal reconstruction by use of implant materials are described. Inspite of a multitude of options and the availability of very different materials none of these methods applied for tracheal reconstruction were successfully introduced into the clinical routine. Essential problems are insufficiencies of anastomoses, stenoses, lack of mucociliary clearance and vascularisation. The advances in Tissue Engineering (TE) offer new therapeutical options also in the field of the reconstructive surgery of the trachea. In pharyngeal reconstruction far reaching developments cannot be recognized at the moment which would allow to give a prognosis of their success in clinical application. A new polymeric implant material consisting of multiblock copolymers was applied in our own work which was regarded as a promising material for the reconstruction of the upper aerodigestive tract (ADT) due to its physicochemical characteristics. In order to test this material for applications in the ADT under extreme chemical, enzymatical, bacterial and mechanical conditions we applied it for the reconstruction of a complete defect of the gastric wall in an animal model. In none of the animals tested either gastrointestinal complications or negative systemic events occurred, however, there was a multilayered regeneration of the gastric wall implying a regular structured mucosa. In future the advanced stem cell technology will allow further progress in the reconstruction of different kind of tissues also in the field of head and neck surgery following the principles of Tissue Engineering.

The effect of radiographic contrast media on the morphology of human erythrocytes.

Echinocyte formation is associated with a rigidification of the cells that possibly affects capillary diffusion and, consequently, the tissue's oxygen supply. This study examines how many echinocytes appeared after the addition of various concentrations of radiographic contrast media (RCM) (Iodixanol 320, Iohexol 350, Iopromide 370, Iomeprol 350 and Iomeprol 400 mg Iodine/ml) compared to red blood cells in isotonic saline solution as well as in autologous plasma. Isotonic saline solution, Iodixanol, Iohexol, Iomeprol 350, Iomeprol 400 and Iopromide in concentrations of 10%, 20% or 40% were added to the plasma of six healthy subjects. Subsequently, the erythrocytes were resuspended in these RCM/plasma mixtures, incubated for 5 minutes at 37 degrees C and then examined under the microscope.The various mixtures and concentrations of the RCM in the mixture all had a significant effect on the number of discocytes (p<0.0001). The percentage of discocytes for all concentrations significantly depended on the RCM/plasma mixture (p=0.0097). Of all the RCM/plasma mixtures used as well as of the NaCl/plasma mixtures, the Iodixanol/plasma mixture showed the most similar discocyte fraction compared to red blood cells in the autologous plasma. At the same time, while Iodixanol in this respect differed from all other RCMs, the other RCMs only differed little from one another with respect to the discocyte fraction.

The effect of radiographic contrast media on the morphology of human venous endothelial cells.

Radiographic contrast media (RCM) can affect the morphology of red blood cells in very different ways but research on how they affect endothelial cell morphology is rudimentary. The effect of two conventional RCMs on human umbilical venous cells over the short term was studied in vitro under static conditions. Cell circumference length, the number of dissolved cell contacts and the number of denuded subendothelial matrix areas were interactively quantified by a computer imaging system after histochemical processing. 1.5 minutes after RCM exposure a significant effect of both RCMs on cell circumference length (CCL) compared to the control cells was evident (p=0.0001 each). The increase after iodixanol was larger than after iomeprol (p=0.0087). After five minutes of exposure, the CCL of exposed cells were significantly larger than those of control cells (p<0.0001 each). The CCL after exposure hardly differed anymore at that time (iomeprol/iodixanol: p=0.0547), though cells exposed to iomeprol tended to be bigger. After both iomeprol (p<0.0001) and iodixanol (p=0.0018), the number of dissolved cell contacts (DCC) increased compared to the control cells. The increases after either RCM were similar (p=0.9633). After five minutes of RCM exposure, the number of DCC was significantly higher than for the control cells (control/iomeprol: p<0.0001; control/iodixanol: p=0.0012). After exposure to iodixanol, significantly fewer DCC were recorded than after iomeprol (p=0.0018). At 1.5 minutes after RCM exposure, the number of denuded subendothelial matrix areas (DSMA) in the cell layer increased both after iomeprol (p<0.0002) and after iodixanol (p=0.0002) compared to the control cells. The increases with the two RCMs were similar (p=0.8618). After five minutes of exposure, the number of DSMA in the cell layer was significantly higher than for the control cells (control/iomeprol: p<0.0001; control/iodixanol: p=0.0015). However, after iodixanol significantly fewer DSMA were recorded than after iomeprol (iomeprol/iodixanol: p=0.0353). The number of dissolved cell/cell contacts and the number of denuded subendothelial matrix areas in the confluent endothelial layer were significantly greater after exposing the endothelial cells for five minutes to iomeprol than after iodixanol.

Determination of the tissue distribution and excretion by accelerator mass spectrometry of the nonadecapeptide 14C-Moli1901 in beagle dogs after intratracheal instillation.

Moli1901 is a 19 residue polycyclic peptide antibiotic which increases chloride transport and water mobilization in airway epithelium. These properties suggest that it may be a useful treatment for cystic fibrosis (CF). In this study, we used accelerator mass spectrometry (AMS) to quantify Moli1901 following administration of only 0.045 microCi of 14C-Moli1901 per dog. Limits of quantitation of AMS were 0.03 (urine) to 0.3 (feces) ng equiv. Moli1901/g. Administration of 14C-Moli1901 by intratracheal instillation (approximately 100 microg) into the left cranial lobe of the lung of beagle dogs resulted in retention of 64% of the dose in the left cranial lobe for up to 28 days. Whole blood and plasma concentrations of 14C were <5 ng/ml at all times after the dose. Concentrations of 14C in whole blood and plasma declined over the first day after the dose and rose thereafter, with the rise in plasma concentrations lagging behind those in whole blood. During the first 3 days after the dose, plasma accounted for the majority of 14C in whole blood, but after that time, plasma accounted for only 25-30% of the 14C in whole blood. Tissue (left and right caudal lung lobe, liver, kidney, spleen, brain) and bile concentrations were low, always less than 0.25% the concentrations found in the left cranial lung lobe. Approximately 13% of the dose was eliminated in urine and feces in 28 days, with fecal elimination accounting for about 10% of the dose. The data presented here are consistent with that obtained in other species. Moli1901 is slowly absorbed and excreted from the lung, and it does not accumulate in other tissues. Moli1901 is currently in the clinic and has proven to be safe in single dose studies in human volunteers and cystic fibrosis patients by the inhalation route. No information on the disposition of the compound in humans is available. This study in dogs demonstrates the feasibility of obtaining that information using 14C-Moli1901 and AMS.

Expression of MMPs and TIMPs in primary epithelial cell cultures of the upper aerodigestive tract seeded on the surface of a novel polymeric biomaterial.

INTRODUCTION: Using standard cell biological and biochemical experimental approaches we were able to test the ability of a particular polymer construct to support the adhesion, proliferation, and the cellular acitivity of pharyngeal cells. The delicate balance between Matrix Metalloproteinases (MMPs) and their endogenous inhibitors (Tissue Inhibitor of MMPs, TIMPs) have a decisive function in the remodeling of the extracellular matrix during cellular ingrowth. Novel polymeric biomaterials may be useful to develop new therapeutic options in head and neck surgery. METHODS: Primary cell cultures of the pharynx of Sprague-Dawley rats were seeded on the surface of a thermoplastic multi-block copolymer and on a polystyrene surface as control. Conditioned media of the primary cells was analyzed for MMPs and TIMPs. The MMP and TIMP expression was analysed by zymography and a radiometric enzyme assay. RESULTS: No statistically significant differences in the levels of MMP-1, MMP-2, MMP-9 and TIMPs were detected between cells grown on the novel polymer surface versus control. CONCLUSION: An appropriate understanding of the molecular machinery that regulates gene expression and cellular growth in tissue engineered constructs is the requirement for an optimal adaptation of biodegradable biomaterials to develop new therapeutic options in otolaryngology and head and neck surgery.

[Cell proliferation and cellular activity of primary cell cultures of the oral cavity after cell seeding on the surface of a degradable, thermoplastic block copolymer]. / Zellproliferation und zelluläre aktivität von primärzellkulturen der mundhöhle nach oberflächenbesiedlung eines abbaubaren, thermoplastischen blockcopolymers.

Using standard cell biological and biochemical methods we were able to test the ability of a degradable, thermoplastic block copolymer to support the adhesion, proliferation, and the cellular activity of primary cell cultures of the oral cavity in vitro. The delicate balance between a group of endogenous enzymes, Matrix Metalloproteinases (MMPs), and their inhibitors (Tissue Inhibitor of MMPs, TIMPs) have a decisive function in the remodeling of the extracellular matrix during processes like wound healing or the integration of biomaterials in surrounding tissues after implantation. Recently developed, biodegradable thermoplastic elastomers with shape-memory properties may be the key to develop new therapeutical options in head and neck surgery. Primary cell cultures of the oral cavity of Sprague-Dawley rats were seeded on the surface of a thermoplastic block copolymer and on a polystyrene surface as control. Conditioned media of the primary cells were analyzed for MMPs and TIMPs after different periods of cell growth. The MMP and TIMP expression was analysed by zymography and a radiometric enzyme assay. No statistically significant differences in the appearance and the kinetic of MMP-1, MMP-2, MMP-9 and TIMPs were detected between cells grown on the polymer surface compared to the control. An appropriate understanding of the molecular processes that regulate cellular growth and integration of a biomaterial in surrounding tissue is the requirement for an optimal adaptation of biodegradable, polymeric biomaterials to the physiological, anatomical, and surgical conditions in vivo to develop new therapeutic options in otolaryngology and head and neck surgery.

Kinetics of conjugation and oxidation of nitrobenzyl alcohols by rat hepatic enzymes.

Previous work has suggested that quantitative differences in the in vitro and in vivo metabolism of mononitrotoluene isomers are a result of differences in the hepatic conjugation and oxidation of the first metabolic intermediates, the mononitrobenzyl alcohols. We have determined the steady-state kinetic parameters, Vmax, Km and V/K, for the metabolism of the nitrobenzyl alcohols by rat hepatic alcohol dehydrogenase, glucuronyltransferase, and sulfotransferase. 3-Nitrobenzyl alcohol was the best substrate for cytosolic alcohol dehydrogenase (Vmax = 1.48 nmoles/min/mg protein, V/K = 3.15 X 10(-3) nmoles/min/mg protein/microM, Km = 503 microM). Vmax and Km values for 4-nitrobenzyl alcohol were similar, but V/K was about 60% of that for 3-nitrobenzyl alcohol. 2-Nitrobenzyl alcohol was not metabolized by the alcohol dehydrogenase preparation used here, but it was metabolized to 2-nitrobenzoic acid by a rat liver mitochondrial preparation. 2-Nitrobenzyl alcohol was the best substrate for microsomal glucuronyltransferase (Vmax = 3.59 nmoles/min/mg protein, V/K = 11.28 X 10(-3) nmoles/min/mg protein/microM, Km = 373 microM). The Vmax for 3-nitrobenzyl alcohol was similar, but the V/K was about half and the Km was about twice that for 2-nitrobenzyl alcohol. The Vmax for 4-nitrobenzyl alcohol was about 40% and the V/K was about half that for 2-nitrobenzyl alcohol. The best substrate for cytosolic sulfotransferase was 4-nitrobenzyl alcohol (Vmax = 1.69 nmoles/min/mg protein, V/K = 37.21 X 10(-3) nmoles/min/mg protein/microM, Km = 48 microM). The Vmax values for the other two benzyl alcohols were similar, but the V/K and Km values were about 11 and 400%, respectively, of those for 4-nitrobenzyl alcohol. These data are in qualitative agreement with results obtained when the nitrobenzyl alcohols were incubated with isolated hepatocytes, but they do not allow quantitative modeling of the data from hepatocytes.

Analytical approaches to the study of the disposition of myelotoxic agents.

A correlation of myelotoxic effect with concentration or a foreign compound of its metabolite at the site of action may provide useful insights into the mechanism of toxic action and/or its amelioration. This correlation requires sensitive and specific assay methods. This communication describes useful methods for the study of benzene disposition in rodents. The assays are sensitive, specific, and rapid. They rely on gas chromatography-mass spectrometry and on high performance liquid chromatography. These methods have allowed measurement of catechol, phenol, and hydroquinone in samples of rodent bone marrow following inhalation exposure to benzene. Their application to the study of benzene metabolism in rat bone marrow in situ is also described.

Cell specificity in DNA binding and repair of chemical carcinogens.

Many animal models for organ specific neoplasia have been developed and used to study the pathogenesis of cancer. Morphologic studies have usually concentrated on the response of target cells, whereas biochemical investigations have usually employed whole organ homogenates. Since hepatocytes comprise nearly 90% of the liver's mass and 70-80% of its DNA, alterations in DNA replication, covalent binding and DNA repair of nonparenchymal cells are usually obscured when whole organ homogenates are used. By utilizing cell separation methods, we have been able to demonstrate differences between hepatocyte and nonparenchymal cell replication. DNA damage and repair following exposure to a variety of hepatocarcinogen. Differences in removal of simple O6-alkylguanine and DNA replication correlate with cell specific carcinogenesis of simply alkylating agents. For several other procarcinogens, including 2-acetylaminofluorene and dinitroluene, cell specificity appears to reside primarily in the differential metabolic competence of hepatocytes and nonparenchymal cells. This results in greater covalent binding of the carcinogen to hepatocyte DNA, although the DNA adducts are removed at a similar rate in both cell types.

Distribution of polybrominated biphenyls after dietary exposure in pregnant and lactating rats and their offspring.

Female rats were fed PBBs in the diet (50 ppm) from day 8 of gestation to day 21 of gestation, from day 1 postpartum to day 14 postpartum or from day 8 of gestation through day 14 postpartum. Levels of PBBs were measured in various tissues. Small concentrations of PBBs (less than 5 microgram/g) were found in the brain, heart, lung, liver, small intestine, placenta, and gravid uterus. Larger concentrations (less than 30 microgram/g) were found in kidneys, the nongravid uterus, skin, mammary tissue, and fat. Lactation did not significantly alter the concentrations of PBBs found in tissues other than mammary tissue. Offspring were subjected to several exposure regimens by cross-fostering. Concentrations of PBBs in the neonatal livers were higher than in the adults nursing them. Transfer of PBBs via the milk appears to be much more important to appearance of PBBs in newborns than does placental transfer.

Comparison of the clinical performance of the Acuvue disposable contact lens and CSI lens in patients with giant papillary conjunctivitis.

Anecdotal reports have suggested that the use of disposable contact lenses is an effective treatment for giant papillary conjunctivitis. In a prospective randomized double-masked study, we compared the clinical performance of the Acuvue (Vistakon, Jacksonville, Florida) disposable contact lens to the traditional daily-wear lens of choice, CSI (Pilkington Barnes Hind, Sunnyvale, California), in 37 patients with previously diagnosed giant papillary conjunctivitis who were examined at one-month intervals for six months. A survey questionnaire for grading symptoms disclosed significant improvement in itching, discharge, and blurred vision in both groups early in the study. Evaluation of patient preference showed that the CSI lens was strongly preferred with regard to lens handling (76% vs 8%). In contrast, the Acuvue lens was strongly preferred with regard to lens comfort (62% vs 11%). For the variable of overall lens preference, there was no significant difference between the two lenses (CSI, 43% and Acuvue, 41%). Multiple regression analysis disclosed that both lens comfort (P < .002) and lens handling (P = .05) contributed significantly to the choices made regarding the dependent variable of overall lens preference. Lens comfort (beta = .71) was observed to be approximately 50% more powerful than lens handling (beta = .48). There was no significant difference in the rate or cost of torn lenses between groups, suggesting that the Acuvue lens can withstand the physical manipulation associated with daily-wear use for up to one month. The results of this study suggest that the use of disposable contact lenses for the treatment of giant papillary conjunctivitis is at least as effective as the traditional daily-wear lens of choice.

Relationship between red blood cell uptake and methemoglobin production by nitrobenzene and dinitrobenzene in vitro.

Nitrobenzene increases methemoglobin formation when incubated with native hemoglobin but not when incubated with red blood cell suspensions. These experiments were designed to determine if transport of nitrobenzene across the red blood cell membrane is a limiting factor for methemoglobin production by red blood cell suspensions. Incubation of [14C]-m-, o- or p-dinitrobenzene, but not mononitrobenzene, with red blood cell suspensions caused a time-dependent increase in methemoglobin. All three dinitrobenzenes and mononitrobenzene crossed the red blood cell membrane and accumulated in the erythrocytes after only 1 min of incubation. Incubation of mononitrobenzene with hemolysates did not result in methemoglobin production. Incubation of red blood cells with the dinitrobenzenes or mononitrobenzene for 1 and 10 min at 4 degrees C did not influence red blood cell uptake of the nitrobenzenes, suggesting that these compounds do not enter the red blood cell by an active process. Dinitrobenzene-induced methemoglobin production was markedly inhibited at 4 degrees C, and may be a result of decreased interaction with hemoglobin and/or decreased metabolism to reactive intermediates which mediate methemoglobin production. These data indicate that red blood cell transport of nitrobenzene is not the limiting factor in methemoglobin production in vitro.

Changes in insulin, glucose and GH concentrations in fed chickens.

The concentrations of insulin, glucose and growth hormone were examined in chickens during the fed and fasted states. In broiler chickens between 5 and 7 weeks of age, blood samples were drawn at intervals which reflected fed (-4 hr), fasted (+0 min) and post meal states (+15 min, immediately after a 15 min meal (and +30 min). Plasma was assayed using a chicken insulin standard in a heterologous RIA. Overall insulin averages, reflecting averaged values for 10 birds/day and 5 replications revealed a decrease with fasting and an increase post-meal. Insulin levels returned to pre-fasted concentrations within 15 min of meal termination. Changes in glucose concentrations correlated with those of insulin with the exception of the +30 min period. When insulin levels were observed in chickens where no food was consumed, insulin levels at +0 and +15 min were not different. No difference in GH concentration occurred at -4 hr, +0 or +15 min but the +30 min value increased (p less than 0.05).

Macromolecular covalent binding of [14C]nitrobenzene in the erythrocyte and spleen of rats and mice.

Nitrobenzene exposure is known to produce red blood cell damage as well as engorgement and sinusoidal congestion of the spleen in male Fischer-344 (F-344) rats but not in male B6C3F1 mice. These studies were conducted to investigate the species differences in the covalent binding of [14C]nitrobenzene in the erythrocyte and spleen and to assess the contribution of nitrobenzene-induced erythrocytic damage to the splenic effects. Total and covalently bound 14C concentrations in erythrocytes of rats were 6-13 times greater than those of mice following a single oral dose of 75, 150, 200 or 300 mg/kg [14C]nitrobenzene, suggesting that species differences in nitrobenzene-induced red blood cell toxicity may be related to differences in erythrocytic accumulation of nitrobenzene and its metabolites. Covalently bound 14C in erythrocytes of rats peaked 24 h following administration of 200 mg [14C]nitrobenzene/kg; in contrast, bound radiolabel in erythrocytes from mice plateaued at 10 h. Splenic engorgement increased in a time-related manner in treated rats but not in mice. Species specificity was also observed in the accumulation of bound radiolabel in the spleen. Gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of lysed, dialyzed erythrocytes from treated rats revealed that hemoglobin was the primary, if not the exclusive, site of macromolecular covalent binding following nitrobenzene treatment. SDS-PAGE of dialyzed rat spleens revealed that 82% of total bound 14C migrated identically to hemoglobin. These data indicate that covalent binding of [14C]nitrobenzene and its metabolites in the spleen is primarily derived from bound 14C from scavenged erythrocytes. Therefore, the species differences in splenic engorgement and accumulation of [14C]nitrobenzene may be related to differences in susceptibility to nitrobenzene-induced red blood cell damage.

Functional and biochemical disposition of 7,12-dimethylbenz[a]anthracene in murine lymphoid cells.

The metabolism of the polycyclic aromatic hydrocarbon (PAH) 7,12-dimethylbenz[a]anthracene (DMBA) was studied in murine lymphocytes. This carcinogen has previously been shown to be immunosuppressive to lymphocytes regardless of their ability to be induced via the Ah locus and receptor. Experiments were designed to quantify the generation of metabolites of DMBA by lymphocytes incubated with [14C]DMBA and to ascertain whether radioactivity was covalently bound to cellular macromolecules in DMBA-exposed lymphocytes. No significant metabolism of DMBA was detected in culture supernatants, except when cultures were incubated in the presence of Arochlor-induced rat liver 9000 x g supernatants (S9). Covalent binding of 14C to cellular macromolecules was enhanced approximately eightfold in the presence of S9. Inhibition of monooxygenase activity by alpha-naphthoflavone did not modulate the immunosuppressive character of DMBA. Furthermore, addition of S9 did not amplify or ablate DMBA-mediated suppression of lymphocyte proliferation to the mitogen concanavalin A (Con A). Selected metabolites of DMBA were evaluated for immunosuppressive effects in cultures stimulated with mitogens and cellular alloantigens. 7-Hydroxymethyl-12-methylbenz[a]anthracene (OHMe) and 5,6-dihydro-5,6-dihydroxybenz[a]anthracene (Diol) were found to cause only slightly greater suppression of lymphocyte responses than DMBA. Thus, it appears that metabolites of DMBA were not responsible for the immunosuppression observed in lymphocyte cultures and that lymphocytes were not equipped to metabolize any significant amount of DMBA. These data lend support to the hypothesis that parent compound alone is responsible for the immunosuppressive effects observed in murine lymphocyte culture.

Hepatic macromolecular covalent binding of mononitrotoluenes in Fischer-344 rats.

The mononitrotoluenes are important industrial chemicals which display isomeric specificity in their ability to induce hepatic DNA excision repair in Fischer-344 rats. Covalent binding of the structurally related hepatocarcinogen, 2,6-dinitrotoluene, to hepatic DNA is markedly decreased by prior administration of the sulfotransferase inhibitors pentachlorophenol (PCP) and 2,6-dichloro-4-nitrophenol (DCNP). The objectives of this study were to determine whether hepatic macromolecular covalent binding of the mononitrotoluene isomers differed and to determine whether covalent binding of the mononitrotoluenes to hepatic DNA in vivo was decreased by inhibitors of sulfotransferase. Male Fischer-344 rats were given a single oral dose of [ring-U-14C]-2-, 3- or 4-nitrotoluene (2-, 3- or 4-NT) and killed at various times thereafter. Livers were removed and analyzed for total and covalently bound radiolabel. Maximal concentrations of total radiolabel were observed between 3 and 12 h after the dose, and there were no large differences among the 3 isomers in peak concentrations achieved. Covalent binding to hepatic macromolecules was maximal 12 h after administration for all three isomers. Thereafter, concentrations of covalently bound 2-NT-derived material were always 2-6 times higher than those of 3- or 4-NT-derived material. When DNA was isolated from livers of rats given the mononitrotoluenes 12 h previously, only 2-NT was observed to covalently bind at concentrations above the limits of detection of the assay. The covalent binding of 2-NT, but not that of 3- or 4-NT, to both total hepatic macromolecules and DNA was markedly decreased by prior administration of either PCP or DCNP. Covalent binding to hepatic DNA was decreased by greater than 96%. The results of this study correlate well with studies which have demonstrated that 2-NT, but not 3- or 4-NT, induces DNA excision repair. Furthermore, they suggest that 2-NT, like the hepatocarcinogen 2,6-dinitrotoluene, requires the action of sulfotransferase for its conversion to a species capable of covalently binding to hepatic DNA.

Diminished drug metabolism due to hormonal effects of cobalt mesoporphyrin on flavoproteins.

Substituted metalloporphyrins, in addition to their use as pharmacological agents, are used to investigate metabolic pathways by inhibiting cytochrome P-450. We have examined the specificity of this approach with cobalt mesoporphyrin (CoMP). In vivo, CoMP (50 mumol/kg, s.c.) decreased rat hepatic microsomal cytochrome P-450, NADPH cytochrome P-450 reductase, benzphetamine N-demethylase (BZPH) activity, and thyroid hormones by > 50%, all of which returned to control levels after 45 days; testosterone levels were also reduced at this dose. The half-life of CoMP was 18 days, which is consistent with this sustained effect. At 10 mumol/kg of CoMP, the reductase activity was decreased, but cytochrome P-450 was unchanged. An effect of residual CoMP on the reductase was ruled out as the CoMP content of tissue fractions was not high enough to inhibit directly the reductase activity (even after 50 mumol CoMP/kg). However, immunoblots indicated a lower level of immunoreactive reductase protein following treatment. After 8 weekly doses of 1 mumol CoMP/kg, BZPH activity was 39% less than control but neither P-450 content nor reductase activity was significantly changed. The P-450 content and reductase activity in rabbits were much less affected by CoMP, perhaps due to differences in the disposition of CoMP. Thyroidectomy decreased reductase activity in rats to an extent that was seen with CoMP at 50 mumol/kg; CoMP treatment of thyroidectomised rats did not further decrease reductase activity. Supplementation with thyroid hormone blocked the CoMP-related decrease. The flavin-containing monooxygenase was decreased by CoMP and by castration, and the decrease was not blocked by the thyroid hormone supplement. Thus in the rat, the CoMP-related decreases in thyroid hormone and testosterone decrease flavoproteins that support or mediate monooxygenase activities. This is contrary to the reported specificity of this class of compound as inhibitors of cytochrome P-450.

Metabolism and toxicity of dinitrobenzene isomers in erythrocytes from Fischer-344 rats, rhesus monkeys and humans.

The metabolism of dinitrobenzene (DNB) isomers in Fischer-344 rat, rhesus monkey and human erythrocytes was investigated. Erythrocytes from all species metabolized o-DNB and p-DNB to S-(nitrophenyl)glutathione conjugates although there were species differences in the rate and extent of conjugate formation. No metabolites of m-DNB were detected in the erythrocytes of any species. The rank order of the ability of the DNB isomers to produce methemoglobin in vitro varied from species to species, but p-DNB was always the most effective isomer. The data suggest that although the erythrocyte can conjugate DNB isomers with glutathione, this pathway offers no substantial protection from methemoglobinemia induced by dinitrobenzenes.