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 significance of augmented radiocolloid uptake by the spleen in patients with malignant melanoma.

Increase in splenic uptake of Tc-99m sulfur colloid was noted in 47 of 147 (32%) patients with cutaneous malignant melanoma early in the coure of disease. Patients with disseminated disease and/or clinical or laboratory evidence of hapatic dysfunction were excluded from study. Recurrence rate of 2 yr was higher for those patients with splenic scans demonstrating augmented uptake compared with patients having normal scans, 36% against 16% (p less than 0.02). These differences resulted from a much more favorable prognosis in women with normal scans contrasted with women with increased uptake, 6% against 26% (p less than 0.05). Women with increased splenic uptake, and all men regardless of scan status, seemed to have a higher rate of recurrence than women with normal spleen scans. Scan status may be an adjunctive prognostic marker in women.

Acute nephrotoxicity induced by isomeric dichloroanilines in Fischer 344 rats.

Chlorinated anilines are widely used as chemical intermediates in the manufacture of numerous dyes, pesticides, drugs and industrial compounds. The purpose of this study was to examine the nephrotoxic potential of the six dichloroaniline (DCA) isomers in vivo and in vitro. In the in vivo studies, male Fischer 344 rats (4-8 rats/group) were administered a single, intraperitoneal injection of a DCA isomer (0.4, 0.8 or 1.0 mmol/kg) as the hydrochloride salt or given vehicle (0.9% saline, 2.5 ml/kg), and renal function monitored at 24 and 48 h. Renal effects induced by DCA were characterized by decreased urine volume, increased proteinuria, hematuria, modest elevations in blood urea nitrogen (BUN) concentrations, decreased accumulation of p-aminohippurate (PAH) by renal cortical slices, and no change or a slight decrease in kidney weight. Renal morphological changes were observed as proximal tubular necrosis with lesser effects on distal tubular cells and collecting ducts. Based on the overall effects on renal function and morphology, the decreasing order of nephrotoxic potential was found to be 3,5-DCA greater than 2,5-DCA greater than 2,4-, 2,6- and 3,4-DCA greater than 2,3-DCA. The ability for the DCA to induce nephrotoxicity correlated well with the lipophilic properties of the DCA isomers and Hammett constants (sigma) for the various chloro substitutions. In the in vitro studies, renal cortical slices from naive male Fischer 344 rats were co-incubated with a DCA isomer (0-10(-3) M) and PAH or tetraethylammonium (TEA). All DCA isomers decreased PAH and TEA accumulation at 10(-3) M DCA concentration in the media with 3,5-DCA inducing the largest decrease at this concentration. These results indicate that DCA are capable of altering renal function in vivo and in vitro and that 3,5-DCA possesses the greatest nephrotoxic potential in vivo and in vitro.

The effect of probenecid on acute N-(3,5-dichlorophenyl)succinimide-induced nephrotoxicity in the Fischer 344 rat.

N-(3,5-Dichlorophenyl)succinimide (NDPS), an experimental agricultural fungicide, has been shown to produce selective nephrotoxicity in rats. Previous studies have shown that a metabolite(s) of extrarenal origin contributes to acute NDPS-induced nephrotoxicity. The purpose of this study was to determine if the organic acid transport inhibitor probenecid could modify the renal toxicity produced by NDPS administration. Male Fischer 344 rats were administered a single intraperitoneal (i.p.) injection of probenecid (60, 90 and 120 mg/kg) or 0.9% saline (1.0 ml/kg) followed 30 min later by NDPS (0.4 or 1.0 mmol/kg, i.p.) or sesame oil (2.5 ml/kg, i.p.) Renal function was monitored at 24 h and 48 h. Probenecid (60 mg/kg) did not markedly alter NDPS-induced renal effects on either post-treatment day. However, pretreatment with probenecid (90 or 120 mg/kg) blocked or attenuated the diuresis, increased proteinuria, decreased tetraethylammonium (TEA), uptake, elevation in blood urea nitrogen (BUN) concentration and increased kidney weight produced by NDPS (0.4 mmol/kg) administration. Only increased kidney weight and BUN concentration, and decreased lactate-stimulated p-aminohippurate (PAH) uptake were altered by probenecid (120 mg/kg) pretreatment when NDPS (1.0 mmol/kg) was given. NDPS-induced changes in renal morphology were not prevented by pretreatment with any probenecid dose. These results suggest that at least one nephrotoxic metabolite of NDPS is an organic acid. However, this acidic metabolite might not be the major nephrotoxic metabolite or a precursor to the major nephrotoxic metabolite(s). The identity of these metabolites remains to be determined.

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.

Trichloroaniline effects on renal function in vivo and in vitro.

Previous studies have demonstrated that mono- and dichloroanilines are capable of inducing acute renal failure in vivo and altering organic ion accumulation by renal cortical slices in vitro. The purpose of this study was to determine the nephrotoxic potential of 4 trichloroaniline (TCA) isomers in vivo and to examine their effects on organic ion accumulation in vitro. In the in vivo experiments, male Fischer-344 rats (4 rats/group) were administered a TCA (0.8 or 1.5 mmol/kg) intraperitoneally (i.p.) or vehicle (sesame oil, 2.5 ml/kg). Renal function was monitored at 24 and 48 h. None of the TCA isomers induced marked renal effects at either time point. In vitro, all TCA isomers were effective in decreasing tetraethylammonium and p-aminohippurate accumulation by renal cortical slices at bath concentrations of 10(-3) M with 2,3,4- and 3,4,5-TCA inducing the greatest reductions. These results indicate that TCA can have a direct effect on renal function in vitro, but that the isomers tested are less potent nephrotoxicants than the nephrotoxic mono- and dichloroanilines.

Acetone effects on N-(3,5-dichlorophenyl)succinimide-induced nephrotoxicity.

Acetone has been shown to potentiate the toxicity of many halogenated hydrocarbons. The purpose of this study was to determine if acetone could alter the acute nephrotoxicity produced by the experimental fungicide N-(3,5-dichlorophenyl)succinimide (NDPS). Male Fischer 344 rats were administered acetone (1, 5 or 10 mmol/kg) or acetone vehicle (corn oil, 10 mg/kg) orally followed 16 h later by a single intraperitoneal injection of NDPS (0.2 or 0.4 mmol/kg) or NDPS vehicle (sesame oil, 2.5 ml/kg) and renal function was monitored at 24 and 48 h. Acetone (1 or 5 mmol/kg) did not alter NDPS (0.2 mmol/kg)-induced renal effects while acetone (10 mmol/kg) pretreatment attenuated NDPS (0.4 mmol/kg)-induced increases in blood urea nitrogen (BUN) concentration and kidney weight but had no effect on NDPS (0.4 mol/kg)-induced changes in urine volume or content, organic ion accumulation by renal cortical slices or renal morphology. These results suggest that acetone weakly attenuates NDPS-induced nephrotoxicity.

3,5-Dichloroaniline-induced nephrotoxicity in the Sprague-Dawley rat.

The nephrotoxic potential of 3,5-dichloroaniline (DCA) was examined in male Sprague-Dawley rats. Rats were administered DCA (0.4, 0.8 or 1.0 mmol/kg, i.p.), or 0.9% saline (1.0 ml/kg, i.p.), and renal function was monitored at 24 and 48 h. DCA (0.4 mmol/kg) administration did not produce evidence of nephrotoxicity. However, DCA (0.8 mmol/kg) administration decreased urine volume and osmolality, increased proteinuria, elevated the blood urea nitrogen (BUN) concentration and decreased basal and lactate-stimulated p-aminohippurate (PAH) accumulation. Three of 4 rats receiving DCA (1.0 mmol/kg) died prior to 48 h postinjection. Incubation of renal cortical slices with DCA resulted in decreased PAH and tetraethylammonium (TEA) uptake when DCA concentrations of 10(-6) M or greater were used. These results indicate that DCA is nephrotoxic to Sprague-Dawley rats when administered in a dose of 0.8 mmol/kg or higher and is capable of altering organic ion transport in vitro.

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.

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.

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.

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.

Tomographic changes in the temporomandibular joint following arthroscopic surgery with lysis and lavage and eminentia release.

Seventy-one patients (128 joints) who underwent temporomandibular arthroscopies with lysis and lavage, capsular stretch, and release of adhesions and lateral capsular fibrosis were followed for an average of 24 months. Prearthroscopic and postarthroscopic temporomandibular joint tomograms were compared; 77.3% of the temporomandibular joints showed no postoperative changes, and 22.7% of the temporomandibular joints studied showed changes. The majority of these tomographic changes involved increase in condylar flattening and beaking. However, postoperative painful symptoms significantly decreased regardless of the radiographic findings.

In vitro activation of mouse skin protein kinase C by fatty acids and their hydroxylated metabolites.

To understand how dietary fatty acids differentially modulate mouse skin tumorigenesis, the ability of specific fatty acids and their derivatives to activate murine epidermal protein kinase C (PKC) in vitro was investigated. Total PKC from untreated female SSIN mouse skin was partially purified and incubated with specific fatty acids at concentrations up to 300 microM in the presence of Ca2+ and phosphatidylserine. The cis-unsaturated fatty acids tested, ranging from 16:1 to 22:6, stimulated PKC activity in a similar dose-dependent manner with an approximate threefold maximum increase over control. Neither the number of cis-double bonds nor the chainlength of these fatty acids affected their relative ability to activate PKC. trans-Fatty acids, with the exception of linoelaidic acid (t,t-18:2n-6), exhibited about half of the potency of their corresponding cis-isomers in stimulating PKC at the plateau concentration (200 microM) or lower. Substitutions close to the double bond on cis-fatty acids abolished their ability to activate PKC. The hydroxylated metabolites of arachidonic acid (20:4n-6) and linoleic acid (c,c-18:2n-6), i.e., the hydroxyeicosatetraenoic acids (HETE) and hydroxyoctadecadienoic acids (HODE), also activated mouse skin PKC in vitro, but only about half as effectively as did the respective parent fatty acids. The results suggest that both hydroxyl substitution and trans-configuration of HETE and HODE are responsible for their reduced ability to activate PKC. Overall the data suggests that the reduced skin tumor yield observed in mice fed diets high in c,c-18:2n-6 is not likely to be due to differences in the ability of c,c-18:2n-6 or 20:4n-6, or their metabolites, to activate PKC.