The kinetic mechanism of the dual phosphorylation of the ATF2 transcription factor by p38 mitogen-activated protein (MAP) kinase alpha. Implications for signal/response profiles of MAP kinase pathways.

The mitogen-activated protein kinases (MAPKs) are a family of enzymes conserved among eukaryotes that regulate cellular activities in response to numerous external signals. They are the terminal component of a three-kinase cascade that is evolutionarily conserved and whose arrangement appears to offer considerable flexibility in encompassing the diverse biological situations for which they are employed. Although multistep protein phosphorylation within mitogen-activated protein kinase (MAPK) cascades can dramatically influence the sensitivity of signal propagation, an investigation of the mechanism of multisite phosphorylation by a MAPK has not been reported. Here we report a kinetic examination of the phosphorylation of Thr-69 and Thr-71 of the glutathione S-transferase fusion protein of the trans-activation domain of activating transcription factor-2 (GST-ATF2-(1-115)) by p38 MAPKalpha (p38alpha) as a model system for the phosphorylation of ATF2 by p38alpha. Our experiments demonstrated that GST-ATF2-(1-115) is phosphorylated in a two-step distributive mechanism, where p38alpha dissociates from GST-ATF2-(1-115) after the initial phosphorylation of either Thr-69 or Thr-71. Whereas p38alpha showed similar specificity for Thr-71 and Thr-69 in the unphosphorylated protein, it displayed a marked difference in specificity toward the mono-phosphoisomers. Phosphorylation of Thr-71 had no significant effect on the rate of Thr-69 phosphorylation, but Thr-69 phosphorylation reduced the specificity, k(cat)/K(M), of p38alpha for Thr-71 by approximately 40-fold. Computer simulation of the mechanism suggests that the activation of ATF2 by p38alpha in vivo is essentially Michaelian and provides insight into how the kinetics of a two-step distributive mechanism can be adapted to modulate effectively the sensitivity of a signal transduction pathway. This work also suggests that whereas MAPKs utilize docking interactions to bind substrates, they can be weak and transient in nature, providing just enough binding energy to promote the phosphorylation of a specific substrate.

The effects of ellagic acid on arylamine N-acetyltransferase activity in the bacterium Pseudomonas aeruginosa.

Arylamine N-acetyltransferase (NAT) activity in Pseudomonas aeruginosa was inhibited by ellagic acid (EA), a naturally occurring dietary plant phenol. By measuring the acetylation of 2-aminofluorene (2-AF), the NAT activity was determined. In P. aeruginosa ATCC 27853, a NAT activity of 1.37 +/- 0.25 nmol/min/10(10) CFU for intact cell and a NAT activity of 5.92 +/- 0.20 nmol/min/mg protein for cytosolic preparation were measured. EA (ranging from 1 to 0.125 mM) showed a dose-dependent inhibition of NAT activities in the analysis of both intact cell and cytosolic preparations. Enzymatic kinetics were determined and found that EA was a potent non-competitive inhibitor of NAT activity in P. aeruginosa ATCC 27853. EA inhibition of NAT activities in P. aeruginosa ATCC 27853 was time-dependent for at least 4 hrs. These data strongly indicated that EA could suppress NAT activity in P. aeruginosa.

Chemopreventive activity of celecoxib, a specific cyclooxygenase-2 inhibitor, and indomethacin against ultraviolet light-induced skin carcinogenesis.

Epidemiological and dietary studies suggest that nonsteroidal anti-inflammatory drugs (NSAIDs) reduce the risk of colon cancer, possibly through a mechanism involving inhibition of cyclooxygenase (COX)-2, which is overexpressed in premalignant adenomatous polyps and colon cancer. Because ultraviolet light (UV) can induce COX-2 and nonspecific NSAIDs can decrease UV-induced skin cancer, we evaluated the ability of two compounds, celecoxib (a specific COX-2 inhibitor) and indomethacin (a nonspecific NSAID), to block UV-induced skin tumor development in SKH:HR-1-hrBr hairless mice. Mice fed 150 or 500 ppm celecoxib showed a dose-dependent reduction (60% and 89%, respectively) in tumor yield. Indomethacin (4ppm) reduced tumor yield by 78%. Although both acute and chronic UV exposure increased cell proliferation and edema, neither compound reduced these parameters. In contrast, UV-induced prostaglandin synthesis in the epidermis was effectively blocked by both compounds. UV-induced increases in COX-2 expression in skin were also not altered in any of the treatment groups. Similarly, tumors that constitutively express high levels of COX-2 displayed no reduction by treatment with celecoxib or indomethacin. The dramatic protective effects of celecoxib suggests that specific COX-2 inhibitors may offer a way to safely reduce the risk of skin cancer in humans.

The effects of plant phenolics, caffeic acid, chlorogenic acid and ferulic acid on arylamine N-acetyltransferase activities in human gastrointestinal microflora.

The possible effects of naturally occurring plant phenolics, caffeic acid (CA), chlorogenic acid (CGA) and ferulic acid (FA) on arylamine N-acetyltransferase (NAT) activities on human gastrointestinal microflora, Escherichia coli, Klebsiella pneumoniae, Enterobacter aerogenes, Citrobacter koseri and Pseudomonas aeruginosa, were examined. The bacterial NAT activities were determined by HPLC measuring the acetylation of 2-aminofluorene (2-AF). Among all examined bacteria, P. aeruginosa exerted the highest NAT activity while C. koseri possessed the lowest NAT activity. CA, CGA and FA could suppress the bacterial NAT activities dose-dependently both in the intact cell and cytosolic fraction analysis. According to the analysis of kinetic parameters in E. coli and P. aeruginosa, CA, CGA and FA were shown to be potent noncompetitive inhibitors of bacterial NAT activities. For the time course experiment, 4 mM of CA and FA could inhibit bacterial NAT activities for at least 4 hour but 4 mM of CGA could only significantly suppress NAT activity in E. coli for the same reaction time. These results strongly demonstrated that CA, CGA and FA inhibited NAT activities in human gastrointestinal bacteria.

Suppression or elevation of cytosolic phospholipase A2 alters keratinocyte prostaglandin synthesis, growth, and apoptosis.

Prostaglandins and other arachidonic acid (AA) metabolites are synthesized by keratinocytes in response to tumor promoters and are produced at very high levels in tumors. After phorbol ester treatment, AA is hydrolyzed from keratinocytes primarily by the cytosolic form of phospholipase A2 (cPLA2), which exhibited a strong substrate preference for phosphatidylcholine over phosphatidylethanolamine and AA over other fatty acids. Phorbol esters increase cPLA2 activity but not the level of expression. To dissociate increased cPLA2 activity from other phorbol ester effects and thus determine the effects of altered AA release on cell growth, the murine keratinocyte cell line, HEL-30, was stably transfected with the sense or antisense cDNA for cPLA2. The resulting cell lines displayed corresponding over- or underexpression and up to 23-fold differences in cPLA2 activity between them. Phorbol ester caused a 15-fold difference in AA release between sense and antisense transfectants. Prostaglandin E2 levels correlated with AA release levels. The sense transfectants showed an enhanced proliferative capacity, based on increased cell number over time and [3H]thymidine incorporation. The antisense transfectants had significantly (>60%) reduced growth rates, compared with both parental cells and sense transfectants. The extent of apoptosis was determined in tumors from cell lines grown in graft chambers in vivo. The number of apoptotic cells was significantly greater in tumors from the sense transfectants, based on terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling staining, compared with the parental or antisense lines. These data are in agreement with a recent study (M. C. Stern et al., Mol. Carcinog., 20: 137-142, 1997) showing a correlation between increased apoptosis and tumor progression in this model system. These results suggest that the elevated eicosanoid synthesis that is observed in skin carcinomas contributes to the growth and progression of these tumors.

The effect of sulindac on arylamine N-acetyltransferase activity in Pseudomonas aeruginosa.

Arylamine N-acetyltransferase (NAT) activity in Pseudomonas aeruginosa was inhibited by sulindac, a drug proposed for cancer prevention. By using high performance liquid chromatography, the NAT activity for acetylation of 2-aminofluorene (2-AF) was examined. The cytosolic NAT activity in P. aeruginosa ATCC 27853 was 5.32 +/- 0.36 nmol/min/mg of protein. Sulindac displayed a dose-dependent inhibition to cytosolic NAT activity in P. aeruginosa with an IC50 value of about 0.46 mM. The NAT activity measured from intact P. aeruginosa cells was 1.39 +/- 0.12 nmol/min/10(10) CFU and this activity was inhibited by sulindac in a similar dose-dependent fashion with an IC50 value of approximately 0.42 mM. Time-course experiments showed that NAT activity measured from intact P. aeruginosa cells was inhibited by sulindac for at least 3 h. Using standard steady-state kinetic analysis, it was demonstrated that sulindac was a possible noncompetitive inhibitor to NAT activity in P. aeruginosa. The results suggest that sulindac suppressed the NAT activity in P. aeruginosa.

The characteristics of arylamine N-Acetyltransferase in Pseudomonas aeruginosa.

N-Acetyltransferase (NAT), responsible for bioactivation and detoxification of arylamines, has been demonstrated to be widely distributed in many organisms ranging from humans to microorganisms. Using high performance liquid chromatography (HPLC) to analyze NAT activity in bacteria, the authors found that Pseudomonas aeruginosa exhibited high NAT activity with 2-aminofluorene (2-AF) as substrate. Characteristics of this bacterial NAT were further investigated. The N-acetylation catalyzed by this enzyme is an acetyl coenzyme A (AcCoA)-dependent reaction. As the concentration of AcCoA in the reaction mixture was increased, the apparent K(m) and Vmax for 2-AF increased. The K(m) and Vmax were 0.504 +/- 0.056 mM and 31.92 +/- 3.23 nmol/min/mg protein, respectively, for the acetylation of 2-AF with 0.5 mM AcCoA. The optimum pH for the enzyme activity was estimated to be around 8.5. It was active at a temperature range from 5 degrees C to 55 degrees C, with maximum activity at 37 degrees C. The enzyme activity was inhibited by divalent metal ions including Cu++, Fe++, Zn++, Ca++, Co++, Mn++, and Mg++, suggesting that a sulfhydryl group is involved in the N-acetylation activity. The three chemical modification agents, iodoacetamide, phenylglyoxal, and diethylpyrocarbonate, all exhibited a dose-, time-, and temperature-dependent inhibition effect. Preincubation of the NAT with AcCoA provided significant protection against the inhibition of iodoacetamide and diethylpyrocarbonate, but only partial protection against the inhibition of phenylglyoxal. These results indicate that cysteine, histidine, and arginine residues are essential for this bacterial enzyme activity, and the first two are likely to reside on the AcCoA binding site, but arginine residue may be located only near the AcCoA binding site. Our data demonstrate that P. aeruginosa possesses highly active N-acetyltransferase which shares a similar catalytic mechanism as that of higher organisms. These findings are very helpful for further investigating the role of arylamine NAT in this bacterial species.

Rhein affects arylamine N-acetyltransferase activity in Helicobacter pylori from peptic ulcer patients.

Arylamine N-acetyltransferase (NAT) activities with 2-aminofluorene and p-aminobenzoic acid were determined in the bacterium Helicobacter pylori collected from peptic ulcer patients. Cytosols or suspensions of H. pylori with or without specific concentrations of rhein co-treatment showed different percentages of 2-aminofluorene and p-aminobenzoic acid acetylation. The data indicate that there was decreased NAT activity associated with increased levels of rhein in H. pylori cytosols. Inhibition of growth studies from H. pylori demonstrated that rhein elicited dose-dependent bacteriostatic activity in H. pylori cultures: i.e. the greater the concentration of rhein, the greater the inhibition of growth to H. pylori. For the cytosol and intact bacteria examination, the apparent values of Km and Vmax were decreased after co-treatment with 40 microM rhein. This report is the first demonstration of rhein inhibition of arylamine N-acetyltransferase activity and rhein inhibition of growth in the bacterium H. pylori.

Identification and characterization of several forms of phospholipase A2 in mouse epidermal keratinocytes.

The tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) stimulated the release of arachidonic acid (AA) from mouse keratinocytes. A distinct difference was observed between the fatty acid release profile elicited by TPA and other stimuli. These findings led to the investigation of keratinocyte phospholipase A2 (PLA2), which catalyzes the release of sn-2 fatty acids from membrane phospholipids and regulates the production of eicosanoids. We characterized and identified several forms of PLA2 in mouse keratinocytes, a cytosolic or cPLA2 and two secretory or sPLA2s in the membrane. The PLA2 in keratinocyte cytosol is sensitive to heating and acid treatment, while resistant to reducing reagent. The PLA2 in keratinocyte membrane is resistant to heating and acid treatment, while sensitive to reducing reagent. These characteristics suggested the presence of a cPLA2 and at least one type of sPLA2. Inhibitor data further confirmed the identities of these PLA2s. The cPLA2 was activated by TPA, and appeared to be responsible for the majority of the specific release of AA observed in mouse keratinocytes treated with TPA. The calcium ionophore A23187, and 4alpha-TPA did not elicit the selectivity towards AA observed with TPA. The release of linoleic acid (LA) and oleic acid (OA) from A23187- and 4alpha-TPA-treated keratinocytes suggests activation of sPLA2. These activities may be due to the existence of both type I and type II sPLA2, as both were identified by polymerase chain reactions. In conclusion, keratinocytes express several forms of phospholipase A2 that differ in their substrate specificities and mechanisms of activation, resulting in distinct agonist-specific fatty acid release profiles.

Ibuprofen inhibits arylamine N-acetyltransferase activity in the bacteria Klebsiella pneumoniae.

Ibuprofen, one of the nonsteroidal anti-inflammatory drugs, inhibited arylamine N-acetyltransferase activity of Klebsiella pneumoniae both in vitro and in vivo. The NAT activities of Klebsiella pneumoniae were inhibited by ibuprofen in a dose-dependent manner both in vitro and in vivo. In vitro, the NAT activity was 0.675 +/- 0.028 nmol/min/mg of protein for the acetylation of 2-aminofluorene. In the presence of 8 mM ibuprofen, the NAT activity was 0.506 +/- 0.002 nmol/min/mg of protein for the acetylation of 2-aminofluorene. In vivo, the NAT activity was 0.279 +/- 0.016 nmol/min/10(10) colony forming units (CFU) for the acetylation of 2-aminofluorene. In the presence of 8 mM ibuprofen, the NAT activity was 0.228 +/- 0.008 nmol/min/10(10) CFU for the acetylation of 2-aminofluorene. The inhibition of NAT activity by ibuprofen was shown to persist for at least 4 h. For in vitro examination, the values of apparent Km and Vmax were 1.08 +/- 0.05 mM and 9.17 +/- 0.11 nmol/min/mg of protein, respectively, for 2-aminofluorene. However, when 8 mM of ibuprofen was added to the reaction mixtures, the values of apparent Km and Vmax were 1.19 +/- 0.01 mM and 6.67 +/- 0.11 nmol/min/mg of protein, respectively, for 2-aminofluorene. For in vivo examination, the values of apparent Km and Vmax were 1.24 +/- 0.48 mM and 4.18 +/- 1.06 nmol/min/10 x 10(10) CFU, respectively, for 2-aminofluorene. However, when 8 mM of ibuprofen was added to the culture, the values of apparent Km and Vmax were 0.95 +/- 0.29 mM and 2.77 +/- 0.37 nmol/min/mg protein, respectively, for 2-aminofluorene, respectively. This report is the first finding of ibuprofen inhibition of arylamine N-acetyltransferase activity in a strain of Klebsiella pneumoniae.

Evidence for arylamine N-acetyltransferase activity in the bacterium Helicobacter pylori.

N-Acetyltransferase activities with p-aminobenzoic acid and 2-aminofluorene were determined in Helicobacter pylori from gastroduodenal disease patients. The N-acetyltransferase activity was determined using an acetyl CoA recycling assay and high pressure liquid chromatography. The N-acetyltransferase activities from a number of Helicobacter pylori samples were found to be 0.91 +/- 0.12 nmole/min/mg protein for the acetylation of 2-aminofluorene and 0.75 +/- 0.22 nmole/min/mg protein for the acetylation of p-aminobenzoic acid. The apparent K(m) and V(max) values obtained were 1.10 +/- 0.08 mM and 2.34 +/- 0.14 nmol/min/mg protein for 2-aminofluorene, and 0.92 +/- 0.09 mM and 2.08 +/- 0.16 nmol/min/mg protein for p-aminobenzoic acid. The optimal pH value for the enzyme activity was 6.0 for both substrates tested. The optimal temperature for enzyme activity was 37 degrees C for both substrates. The N-acetyltransferase activity was inhibited by iodacetamide: at 0.25 mM iodacetamide, activity was reduced 50% and 1.0 mM iodacetamide inhibited activity more than 90%. Among a series of divalent cations and salts, Cu2+ and Zn2+ were demonstrated to be the most potent inhibitors. Among the protease inhibitors, only ethylenediaminetetraacetic acid significantly protected N-acetyltransferase. Iodoacetic acid, in contrast to the other agents, markedly inhibited N-acetyltransferase. This is the first demonstration of acetyl CoA:arylamine N-acetyltransferase activity in Helicobacter pylori.

Glycyrrhizic acid inhibits arylamine N-acetyltransferase activity in Klebsiella pneumoniae in vitro.

Glycyrrhizic acid, one of the proposed chemopreventive drugs, was used to inhibit arylamine N-acetyltransferase (NAT) activity in Klebsiella pneumoniae, both in cytosol and intact bacteria. The NAT activity was measured by using high-performance liquid chromatography to assay the amounts of 2-acetyl-aminofluorene and remaining 2-aminofluorene. The NAT activity in K. pneumoniae was inhibited by glycyrrhizic acid in a dose-dependent manner. The cytosol NAT activities were 0.675 +/- 0.028 nmol min(-1) mg(-1) protein for the acetylation of 2-aminofluorene without glycyrrhizic acid and 0.367 +/- 0.008 nmol min(-1) mg(-1) protein with 8 mM glycyrrhizic acid. The NAT activities measured from intact bacteria were 0.308 +/- 0.018 nmol min(-1) 10(-10) colony forming units for the acetylation of 2-aminofluorene without glycyrrhizic acid and 0.236 +/- 0.005 nmol min(-1) 10(-10) colony forming units in the presence of 8 mM glycyrrhizic acid. The inhibition of NAT activity by glycyrrhizic acid was demonstrated to remain for at least 4 h. The apparent Km and Vmax values calculated from cytosol NAT were 1.08 +/- 0.05 mM and 9.09 +/- 0.11 nmol min(-1) mg(-1) protein, respectively, for 2-aminofluorene. In the presence of 8 mM glycyrrhizic acid, the apparent Km and Vmax values were 0.15 +/- 0.01 mM and 0.95 +/- 0.11 nmol min(-1) mg(-1) protein, respectively, for 2-aminofluorene. In intact bacteria, the apparent Km and Vmax values were 1.28 +/- 0.48 mM and 4.08 +/- 1.06 nmol min(-1) 10(-10) colony forming units, respectively, for 2-aminofluorene. However, in the presence of 8 mM glycyrrhizic acid, the apparent Km and Vmax values were 0.67 +/- 0.09 mM and 1.82 +/- 0.37 nmol min(-1) 10(-10) colony forming units, respectively, for 2-aminofluorene. Taking these results together, the NAT activity in K. pneumoniae was inhibited by glycyrrhizic acid both in cytosol and intact bacteria. This study provides the first evidence to demonstrate that glycyrrhizic acid inhibits bacterial NAT activity.

The effect of vitamin C on N-acetyltransferase activity in Klebsiella pneumoniae.

This study was designed to assess the effect of vitamin C on arylamine N-acetyltransferase (NAT) activity in Klebsiella pneumoniae by using HPLC to measure the acetylation of 2-aminofluorene (2-AF) with and without vitamin C. Two assay systems were performed, one with intact bacterial cell suspensions, the other with S-9 fractions (9000g supernatant). It was found that vitamin C promoted NAT activity in K. pneumoniae in a dose-dependent manner in both systems. 4 and 8 mM vitamin C were selected for further studies in S-9 fractions and intact cell systems, respectively. Through a 4-hr time course study, vitamin C promoted the N-acetylation of 2-AF in both assay systems, but, the longer the reaction time lasted, the lower the promotion rate. In the kinetic studies, vitamin C increased the value of Km from 0.42+/-0.03 mM to 2.43+/-0.87 mM in S-9 fraction assays and from 0.54+/-0.03 mM to 0.85+/-0.18 mM in intact cell assays. Vitamin C also increased the apparent Vmax values from 3.5 +/-0.08 to 39.66+/-9.81 nmol/min/mg protein in S-9 fraction assays, and from 1.28+/-0.06 to 4.88+/-0.87 nmol/min/10(10) CFU in intact cell assays, for acetylation of 2-AF. In the presence of vitamin C, the NAT activity was increased from 0.58+/-0.06 to 1.34+/-0.02 nmol/min/mg protein in S-9 fractions, and from 0.18+/-0.02 to 0.40+/-0.02 nmol/min/10(10) CFU in intact cells, for acetylation of 2-AF. From the present study, it is concluded that vitamin C does promote the N-acetylation of 2-AF in K. pneumoniae. This is a first report suggesting that oral vitamin C may be involved in modifying the mutagenicity/carcinogenicity of ingested arylamines through enhancing the NAT activity of human enteric bacteria. This interaction should be pursued in future in vivo studies.

Arachidonate has protumor-promoting action that is inhibited by linoleate in mouse skin carcinogenesis.

Previous studies demonstrated a requirement for arachidonic acid metabolites in tumor development in mouse skin. The goal of this study was to determine whether the arachidonate content of epidermal phospholipids could be altered by increasing dietary levels of linoleate and whether specific metabolites of linoleate and arachidonate have dissimilar biological effects. In a series of tumor studies in which the quantity of dietary linoleate was incrementally increased, a slight reduction in phospholipid levels of arachidonate was observed that correlated with an increased phospholipid level of linoleate and a suppression in tumor yield. A comparison of the arachidonate lipoxygenase metabolite 12-hydroxyeicosatetraenoic acid (12-HETE) with the 13-hydroxyoctadecadienoic acid (13-HODE) lipoxygenase metabolite of linoleate revealed that 12-HETE has biological activities that mimic the phorbol ester tumor promoters, whereas 13-HODE has antithetical effects. Specifically, 12(S)-HETE enhanced the activation of protein kinase C by phorbol esters, mimicked phorbol ester-induced adhesion of keratinocytes to fibronectin and mimicked phorbol ester repression of expression of a differentiation-related gene, keratin-1. 13-HODE blocked 12-HETE-induced cell adhesion and prevented 12-HETE-induced suppression of keratin-1 expression. Overall, these studies suggest that arachidonate and linoleate have opposing functions in the epidermis, particularly with regard to events involved in tumor development.

3,5-Dichloroaniline toxicity in Fischer 344 rats pretreated with inhibitors and inducers of cytochrome P450.

3,5-Dichloroaniline (3,5-DCA), a derivative needed in the manufacture of dyes, pesticides and industrial compounds has been reported to induce renal damage. This study investigated whether pretreatment with inducers or inhibitors of P450 altered 3,5-DCA toxicity. P450 levels were induced in male Fischer 344 rats (4-12/group) by pretreatment (i.p.) with phenobarbital (PB, 75 mg/kg/day for 3 days), beta-naphthoflavone (BNF, 100 mg/kg/day for 4 days) or pyridine (PYR, 100 mg/kg/day for 4 days). P450 activity was inhibited by pretreatment with piperonyl butoxide (PiBx) 30 min prior to injection of 3,5-DCA. Upon completion of a designated pretreatment regimen, 0.4 or 0.8 mmol/kg 3,5-DCA was injected into F344 rats. Pair-fed controls were injected with 25% ethanol solution or physiological saline (2.5 ml/kg). The renal changes monitored at 24 and 48 h following treatment with 0.8 mmol/kg 3,5-DCA were characterized by increased blood urea nitrogen (BUN) level and decreased renal cortical slice accumulation of p-aminohippurate (PAH). Plasma alanine transaminase activity (ALT/GPT) was increased 24 h after injection of 0.8 mmol/kg 3,5-DCA while liver wt. was unchanged. PB or PYR pretreatment did not alter the renal or hepatic effects of 3,5-DCA while BNF pretreatment slightly reduced toxicity. In contrast, PiBx pretreatment increased the renal and hepatic changes associated with 3,5-DCA. The results with PiBx suggest that either the parent compound possesses some direct cytotoxicity or that a toxic metabolite was generated through a biotransformation pathway not inhibited by PiBx.

Fibrous ankylosis of the temporomandibular joint: report of a case with atypical presentation.

A case of chronic unilateral mandibular dislocation with development of fibrous ankylosis is presented. This was an unusual presentation of intracapsular tissue ankylosis to the eminentia, as well as retrocondylar cicatrix combined with contralateral mandibular osseous compensations and remodeling with resulting ramus impingements upon relocation of the condyle. Various diagnostic and therapeutic considerations are reviewed and discussed.

Effect of chemical form, route of administration and vehicle on 3,5-dichloroaniline-induced nephrotoxicity in the Fischer 344 rat.

Chloroanilines are widely used chemical intermediates for the manufacture of dyes, agricultural chemicals and industrial compounds. Nephrotoxicity occurs as one toxicity following intraperitoneal (i.p.) administration of chloroaniline hydrochlorides to rats. The purpose of this study was to examine the effect of chemical form, route of administration and vehicle on 3,5-dichloroaniline-induced nephrotoxicity. In one set of studies, male Fischer 344 rats (four to eight per group) were administered a single i.p. injection of 3,5-dichloroaniline free base or hydrochloride salt, cysteine hydrochloride or ornithine hydrochloride (0.8, 1.0 or 1.5 mmol kg-1) or an appropriate vehicle and renal function monitored for 48 h. Only 3,5-dichloroaniline hydrochloride induced nephrotoxicity that was characterized as acute renal failure. When 3,5-dichloroaniline free base (0.8 mmol kg-1) was administered in dimethyl sulfoxide (DMSO), all rats died within 24 h. In a second experiment, the free base or hydrochloride form of 3,5-dichloroaniline (1.5 mmol kg-1) or vehicle (0.9% saline or sesame oil, respectively) were administered orally and renal function monitored for 48 h. No evidence of nephrotoxicity was observed following either treatment. However, when the hydrochloride salt was given in 25% DMSO in 0.9% saline, all rats died within 24 h, with two rats demonstrating increased proteinuria, glucosuria and hematuria within the first 6 h after treatment. These results demonstrate that 3,5-dichloroaniline nephrotoxicity is potentiated by the administration of systemic acid, but that acid alone has no effect on renal function at the dose tested. Also, 3,5-dichloroaniline (hydrochloride or free base form) is less toxic orally than when administered i.p.(ABSTRACT TRUNCATED AT 250 WORDS)