Involvement of toxicity as an early event in urinary bladder carcinogenesis induced by phenethyl isothiocyanate,benzyl isothiocyanate,and analogues in F344 rats

Phenethyl isothiocyanate (PEITC)(1) and benzyl isothiocyanate (BITC), naturally occurring constituents of cruciferous vegetables, have been reported to exert inhibitory effects against development of tobacco-specific carcinogen-induced lung tumors and are regarded as promising chemopreventive agents for lung cancer. However, tumor promoting and carcinogenic activities in the rat urinary bladder have been detected in several animal models. The purpose of the present study was to investigate early changes in rat urinary bladder epithelium induced by PEITC and BITC and to explore promotion/carcinogenic mechanisms. In the first experiment, in order to assess acute toxic effects, PEITC or BITC at 0.1% each in the diet were administered to 6-week-old F344 rats for 1, 2, 3, and 7 days and sequential histopathological assessment and urinalysis were performed. In the second and third experiments, structure-activity relationships of PEITC, BITC and 8 other analogues, benzyl isocyanate and benzyl thiocyanate, and phenyl-, alpha-naphthyl-, tert-butyl-, butyl-, methyl-, and ethyl isothiocyanates (ITCs) were explored in a 14-day experiment. In the first experiment, the urinary pH was significantly lowered on day 1 by both PEITC and BITC. Striking features of toxicity, such as marked inflammatory changes characterized by cellular infiltration, apoptosis/single cell necrosis, cytoplasmic vacuolation, erosion, and hemorrhage in the urinary bladder were caused, with peaks apparent on days 2 or 3, respectively. Sequential change in 5-bromo-2'-deoxyuridine (BrdU) labeling indices was in line with the inflammatory response, but the thickness of the urinary bladder epithelium continued to gradually increase up to day 7. In the second and third experiments, simple and papillary or nodular (PN) hyperplasias were observed after 14-days treatment with PEITC, BITC, and phenyl- and butyl ITCs. These results suggest that continuous urinary epithelial cell proliferation due to cytotoxicity may play an important role in the early stage of rat urinary bladder carcinogenesis due to oral administration of ITCs. In addition, hydrophobic activity of ITCs, dependent on the alkyl carbon chain length, might strongly influence the induction of bladder lesions in rats.     

Genotoxic effects of benzyl isothiocyanate, a natural chemopreventive agent

Benzyl isothiocyanate (BITC) is contained in cruciferous plants which are part of the human diet. Numerous reports indicate that BITC prevents chemically induced cancer in laboratory animals and it has been postulated that BITC might also be chemoprotective in humans. On the other hand, evidence is accumulating that this compound is a potent genotoxin in mammalian cells by itself. To further elucidate the potential hazards of BITC, we investigated its genotoxic effects in different in vitro genotoxicity tests and in animal models. In in vitro experiments [differential DNA repair assay with Escherichia coli, micronucleus assay with human HepG2 cells and single cell gel electrophoresis (SCGE) assay with hepatocytes and gastrointestinal tract cells] pronounced dose-dependent genotoxic effects were found at low dose levels (相似文献   

Studies on the disturbance of glucuronide formation in infectious hepatitis

The ability of the liver to form glucuronides was measured in 10 patients with infectious hepatitis. One test was done at the onset and another about four weeks later after the clinical symptoms had disappeared. N-acetyl-p-aminophenol (N.A.P.A.) or acetanilide was administered in doses ranging from 10 to 20 mg. per kg. body weight, either orally or by intravenous injection. N.A.P.A. is conjugated by the liver at the hydroxyl group and excreted in the urine as sulphuric and glucuronic acid conjugates. Total conjugated p-aminophenol, free N.A.P.A., and N.A.P.A. glucuronide were estimated in the urine of our patients. In the blood the disappearance of N.A.P.A. (free form) and the formation of N.A.P.A. glucuronide were traced.     

Investigations of rodent urinary bladder carcinogens: collection, processing, and evaluation of urine and bladders

Examination of the urine and the bladder epithelium are essential to the investigation of mechanisms of urinary bladder carcinogens in rodents. However, urine and bladder tissue specimens must be collected and processed properly if accurate data are to be derived. The optimum specimen for analysis of urinary constituents is fresh void urine collected from nonfasting animals. Fasting the animal prior to urine collection changes the normal composition, including pH. Many of the normal urinary constituents can influence the mode of action of bladder carcinogens, especially for non-genotoxic agents. Light microscopy is routinely used to examine the bladder epithelium. However, it is often necessary to use more sensitive techniques, such as scanning electron microscopy (SEM) to detect subtle cytotoxic changes in the superficial cell layer of the urothelium, and bromodeoxyuridine (BrdU) incorporation, PCNA, or Ki-67 immunohistochemistry to determine the labeling index for cell proliferation. The urinary bladder must be handled gently and inflated with fixative in situ before the animal dies to avoid artifactual autolytic damage to the bladder epithelium that is visible by SEM and may be mistaken for treatment-related changes. The purpose of this paper is to provide information for the proper collection and examination of urine and the urinary bladder.     

Effects of TRPA1 agonists mustard oil and cinnamaldehyde on lumbar spinal wide-dynamic range neuronal responses to innocuous and noxious cutaneous stimuli in rats

Mustard oil [allyl isothiocyanate (AITC)] and cinnamaldehyde (CA), agonists of the ion channel TRPA1 expressed in sensory neurons, elicit a burning sensation and heat hyperalgesia. We tested whether these phenomena are reflected in the responses of lumbar spinal wide-dynamic range (WDR) neurons recorded in pentobarbital-anesthetized rats. Responses to electrical and graded mechanical and noxious thermal stimulation were tested before and after cutaneous application of AITC or CA. Repetitive application of AITC initially increased the firing rate of 52% of units followed by rapid desensitization that persisted when AITC was reapplied 30 min later. Responses to noxious thermal, but not mechanical, stimuli were significantly enhanced irrespective of whether the neuron was directly activated by AITC. Windup elicited by percutaneous or sciatic nerve electrical stimulation was significantly reduced post-AITC. These results indicate that AITC produced central inhibition and peripheral sensitization of heat nociceptors. CA did not directly excite WDR neurons, and significantly enhanced responses to noxious heat while not affecting windup or responses to skin cooling or mechanical stimulation, indicating a peripheral sensitization of heat nociceptors.     

A preliminary physiologically based pharmacokinetic model for naphthalene and naphthalene oxide in mice and rats

Naphthalene is a toxicant with unusual species and tissue specificity that has been the subject ofin vitro studies. We describe a preliminary physiologically based pharmacokinetic (PBPK) model for naphthalene constructed solely fromin vitro data for comparison to animal data without the use of adjustable parameters. The prototypical PBPK model containing five lumped tissue compartments was developed to describe the uptake and metabolism of naphthalene by mice and rats dosed intraperitoneally (ip) and orally (po). The model incorporates circulation and biotransformation of the semistable reactive intermediate, naphthalene oxide, as well as the parent compound naphthalene. Circulation is included because the toxic action of naphthalene has been proposed to be caused by the formation of a reactive metabolite in one organ (liver) and its circulation to another organ (lung) being adversely affected by the metabolite. The model allows conversion of naphthalene oxide into dihydrodiol, glutathione (GSH) conjugates, 1-naphthol (nonenzymatically) and covalently bound adducts with proteins. Model simulations are compared with previously reportedin vivo measurements of glutathione depletion, mercapturic acid formation, and covalently bound protein formation. The mouse model predicts accurately the amount of mercapturates excreted, the effect of various pretreatments, and the extent of covalent binding in the lung and liver resulting from ip administration, including the sharp increase in binding between 200 and 400 mg/kg. This work was supported, in part, by grants from the National Science Foundation (BCS-9016798). Paracelsian, Inc., and the Cornell Biotechnology Program, which is sponsored by the New York State Science and Technology Foundation, a consortium of industries, and the National Science Foundation.     

Urothelial carcinogenesis in the urinary bladder of male rats treated with muraglitazar, a PPAR alpha/gamma agonist: Evidence for urolithiasis as the inciting event in the mode of action

Muraglitazar, a PPARalpha/gamma agonist, dose-dependently increased urinary bladder tumors in male Harlan Sprague-Dawley (HSD) rats administered 5, 30, or 50 mg/kg/day for up to 2 years. To determine the mode of tumor development, male HSD rats were treated daily for up to 21 months at doses of 0, 1, or 50 mg/kg while being fed either a normal or 1% NH4Cl-acidified diet. Muraglitazar-associated, time-dependent changes in urine composition, urothelial mitogenesis and apoptosis, and urothelial morphology were assessed. In control and treated rats fed a normal diet, urine pH was generally > or = 6.5, which facilitates formation of calcium-and magnesium-containing solids, particularly in the presence of other prolithogenic changes in rat urine. Urinary citrate, an inhibitor of lithogenesis, and soluble calcium concentrations were dose dependently decreased in association with increased calcium phosphate precipitate, crystals and/or microcalculi; magnesium ammonium phosphate crystals and aggregates; and calcium oxalate-containing thin, rod-like crystals. Morphologically, sustained urothelial cytotoxicity and proliferation with a ventral bladder predilection were noted in treated rats by month 1 and urinary carcinomas with a similar distribution occurred by month 9. Urothelial apoptotic rates were unaffected by muraglitazar treatment or diet. In muraglitazar-treated rats fed an acidified diet, urine pH was invariably < 6.5, which inhibited formation of calcium-and magnesium-containing solids. Moreover, dietary acidification prevented the urothelial cytotoxic, proliferative, and tumorigenic responses. Collectively, these data support an indirect pharmacologic mode of urinary bladder tumor development involving alterations in urine composition that predispose to urolithiasis and associated decreases in urine-soluble calcium concentrations.     

Rat urinary bladder epithelial lesions induced by acrolein

Acrolein, a constituent of cigarette smoke and a metabolite of cyclophosphamide, has been shown to induce acute cytotoxicity of the rat urinary bladder mucosa when instilled directly into the bladder lumen. To evaluate the effects of systemic administration, we examined the rat urinary bladder following intragastric or intraperitoneal administration of acrolein to male F344 rats. In an initial experiment, acrolein was administered at a dose of 25 mg/kg, which proved to be extremely toxic. Five of 12 rats injected intragastrically and 5 of 12 injected intraperitoneally died within 24 hrs. After 2 days, 3 of the 3 surviving rats injected intraperitoneally had focal simple hyperplasia of the urinary bladder. None of the rats injected intragastrically had bladder hyperplasia. In a second experiment, acrolein was administered by intraperitoneal injection at doses of 0.5, 1, 2, 4, and 6 mg/kg. Five days later, the labeling index of the bladder mucosa was evaluated by autoradiography. In rats injected with 6 mg/kg of acrolein, the labeling index was significantly increased compared to the other doses and compared to a vehicle injection control group. These data indicate that sufficient acrolein reaches the urinary bladder to induce a proliferative response following intraperitoneal administration.     

The enhancing effect of the antioxidant N-acetylcysteine on urinary bladder injury induced by dimethylarsinic acid

Dimethylarsinic acid (DMA(V)), the major excreted metabolite of inorganic arsenic, is carcinogenic to the rat urinary bladder. Oxidative stress has been proposed as one possible mechanism of DMA(V)-induced carcinogenesis. The authors determined whether the antioxidant N-acetylcysteine (NAC) modifies DMA(V)-induced urinary bladder injury in rats. The treatment solutions--DMA(V) at 10 mg/kg, NAC at 90 or 1.6 mg/kg (high or low dose, respectively), and their combination--were intravesically instilled into female F344 rats over two hours under pentobarbital anesthesia. The treatment was conducted twice with an interval of three days. All animals were euthanized one day after the second treatment. NAC (low dose) alone did not induce histopathological changes or increase 5-bromo-2'-deoxyuridine (BrdU) labeling index in urothelial cells. Both DMA(V) and NAC (high dose) induced a weak neutrophil infiltration and an increase in the BrdU labeling index; these pathological changes were enhanced by the combined treatment of DMA(V) and NAC (high or low dose). Increased oxidative stress and urothelial cell hyperplasia with evidence of activated p44/42 MAPK (ERK1/2) and cyclin D1 were found in the DMA(V) and NAC (high dose) cotreated group. These results suggest that cotreatment with NAC enhanced DMA(V)-induced urinary bladder injury and that the effects may be mediated by excess oxidative stress and ERK signaling.     

Evaluation of urinary dihydrocodeine excretion in human by gas chromatography-mass spectrometry

Urinary metabolic pattern after the therapeutic peroral dose of dihydrocodeine tartrate to six human volunteers has been explored. Using the GC-MS analytical method, we have found that the major part of the dose administered is eliminated via urine within the first 24 h. However, the analytical monitoring of dihydrocodeine and its metabolites in urine was still possible 72 h after the dose was administered. The dihydrocodeine equivalent amounts excreted in urine in 72 h ranged between 32 and 108% of the dose, on average 62% in all individuals. The major metabolite excreted into urine was a 6-conjugate of dihydrocodeine, then in a lesser amount a 6-conjugate of nordihydrocodeine (both conjugated to approximately 65%). The O-demethylated metabolite dihydromorphine was of a minor amount and was 3,6-conjugated in 85%. Traces of nordihydromorphine and hydrocodone were confirmed as other metabolites of dihydrocodeine in our study. This information can be useful in interpretation of toxicological findings in forensic practice.     

In vivo metabolism of tritiated LHRH by the whole kidney and individual tubules of rats

[pyroglutamyl-3,4-3H]Luteinizing hormone-releasing hormone ([3H]LHRH) and [14C]inulin were infused into individual nephrons in Inactin-anesthetized rats and the amount of radioactive label and the identity of the radioactively labeled material in urine were determined. The site of infusion was identified by latex injection and microdissection. [3H]LHRH was microinfused at 1.5 X 10(-5 M (concentration 10(6)-10(7) higher than in plasma) and analysis of urinary metabolites was performed by high-performance liquid chromatography. The urinary recovery of tritium label was 81% when proximal tubules were infused and 94% when distal tubules were infused. For proximal tubules 90% of the label recovered in urine appeared as pGlu-His (metabolite 2), pGlu-His-Trp (metabolite 3), and pGlu-His-Trp-Ser (metabolite 4), and 10% as LHRH. With distal tubules only LHRH was detected in the urine. [3H]LHRH was presented to the renal artery of the filtering rat kidney in vivo, and urine and renal venous blood were analyzed for breakdown products. The urine contained metabolites 2, 3, and 4 and no LHRH, whereas venous blood contained mainly pGlu, metabolite 4, and LHRH. When [3H]LHRH was perfused in vivo through the nonfiltering rat kidney or rat lower limb, renal or femoral venous blood was found to contain only LHRH. These studies suggest that [3H]LHRH undergoes glomerular filtration and contact digestion by brush border enzymes of the proximal tubule to produce metabolites 2, 3, and 4. These metabolites and possibly LHRH are partially reabsorbed and undergo further intracellular degradation to produce pGlu. Endothelial and interstitial cells in the kidney and leg do not appreciably metabolize [3H]LHRH.     

Conjugation of acetaldehyde with cysteinylglycine,the first metabolite in glutathione breakdown byγ-glutamyltranspeptidase

Glutathione and its metabolites were examined for reactivity to acetaldehyde. When acetaldehyde was incubated with glutathione alone, there was only a slight decrease of acetaldehyde, while an apparently equimolar reaction between acetaldehyde and free sulfhydryl was observed with the addition of -glutamyltranspeptidase. Cysteinylglycine, the first metabolite in the glutathione breakdown by -glutamyltranspeptidase, showed a rapid and equimolar reactivity to acetaldehyde and such was comparable to the reaction seen withl-cysteine ord-penicillamine. In light of the chemical structure, cysteinylglycine probably conjugates with acetaldehyde to form thiazolidinecarboxylic acid derivatives, 2-methyl-thiazolidine-4-carbonyl-glycine, and if so, the alteration of glutathione metabolism by acetaldehyde during ethanol intoxication warrants further attention.     

Correlations between urinary nicotine or cotinine and urinary mutagenicity in smokers on controlled diets

Cigarette smokers have been reported to void urine that is more mutagenic, as measured in the Salmonella/microsome assay, than urine voided by nonsmokers. Several previous studies have attempted to correlate indices of tobacco smoke exposure (e.g. nicotine, cotinine, tar intake) with urinary mutagenicity, with conflicting results. These studies generally involved small numbers of smokers and did not carefully control diet, which is known to affect urinary mutagenicity markedly. Our objective was to conduct a controlled study to determine clearly if there were a correlation between urinary nicotine, cotinine, or nicotine + cotinine and urinary mutagenicity and to determine if nicotine or its major metabolite plays a role in the mutagenicity of urine from cigarette smokers. We used a large number of smokers (31), each of whom smoked both a tobacco-burning cigarette and a tobacco-heating cigarette on consecutive weeks, and we prepared and served identical diets to all subjects. Nicotine and cotinine concentrations were determined in small aliquots from urine samples collected over 24 hr, and the remaining urine sample was extracted and concentrated on XAD-2 resin for mutagenicity assays in the Salmonella/microsome test. Nicotine, cotinine, and nicotine + cotinine were statistically correlated with mutagenicity of urine from smokers of the tobacco-burning cigarette, but there was no correlation between nicotine, cotinine, or nicotine + cotinine and mutagenicity of urine from smokers of the tobacco-heating cigarettes. Thus, although urinary nicotine and cotinine concentrations correlate with urinary mutagenicity in smokers of tobacco-burning cigarettes, the present results indicate that nicotine and its metabolite are not responsible for the mutagenicity of smokers' urine.     

Recent development of determination methods for adrenocortical hormones and catecholamines

Newly developed resins and high-performance liquid chromatography (HPLC) with column switching technique have enabled on-line solid-phase extraction, purification and derivatization for highly sensitive determination in recently developed determination methods for serum cortisol, urinary 17-ketosteroids (17-KS), urinary conjugated 17-KS fractions and urinary free catecholamines (CA). These methods are (1) determination of serum cortisol by HPLC with a BSA-ODS column (TSK) and serum sample direct injection, (2) determination of fluorescently derivatized serum cortisol with sulfuric acid and ethanol by column switching HPLC, (3) determination of urinary 17-KS with no need of previous hydrolysis of the conjugated forms, (4) HPLC of conjugated 17-KS fluorescently prelabelled with dansyl hydrazine, (5) HPLC of urinary free catecholamines (CA) with on-line extraction using a cation exchange column and on-line fluorescent derivatization with 1,2-diphenylethylenediamine, (6) ion pair HPLC of urinary free CA with on-line extraction using a hard boric acid gel column and postcolumn trihydroxyindole derivatization, and (7) HPLC of urinary free CA with on-line extraction and solid-phase derivatization using a boric acid gel column, o-phthalaldehyde and 2-mercaptoethanol, have achieved no previous or simple sample extraction, more rapid, accurate and higher sensitive determination comparison with ordinary methods for these substances and furthermore, some methods have enabled fully automated analysis.     

Monocytes and neutrophils oxidize low density lipoprotein making it cytotoxic

Free radicals are believed to be involved in leukocyte induced tissue injury. The present studies were performed to determine whether low density lipoprotein (LDL) might serve as a mediator of tissue injury after leukocyte induced free radical oxidation of LDL. Our results show that incubation of LDL with monocytes or polymorphonuclear leukocytes (PMN) leads to oxidation of the lipoprotein rendering it toxic to proliferating fibroblasts. Monocyte activation enhances these effects. Butylated hydroxytoluene (BHT), vitamin E (vit E) and glutathione (GSH) virtually prevent the oxidation of LDL and the formation of cytotoxic LDL, indicating that these alterations are mediated by leukocyte-derived free radicals. This is the first demonstration that short-lived free radicals emanating from phagocytic cells could mediate cell injury through the action of a stable cytotoxin formed by the oxidation of LDL. The fact that lipoproteins can transfer a cytotoxic effect from leukocytes to proliferating cells reveals a pathway for cell destruction which may have implications in atherosclerotic plaque progression, macrophage mediated toxicity to tumor cells and tissue injury by inflammatory processes.     

Inhibitory effect of isothiocyanate derivant targeting AGPS by computer-aid drug design on proliferation of glioma and hepatic carcinoma cells

Lipids metabolism was involved in the process of many types of tumor and alkylglycerone phosphate synthase (AGPS) was considered implicated in tumor process. Benzyl isothiocyanate (BITC) showed the inhibitory effect of tumor and AGPS activity, therefore, we screened a group of small molecular compound based on BITC by computer-aid design targeting AGPS and the results showed that the derivants could suppress the proliferation, the expression of tumor related genes such as survivin and Bcl-2, and the level of ether lipids such as lysophosphatidic acid ether (LPAe) and platelet activating factor ether (PAFe); however, the activity of caspase-3/8 was improved in glioma U87MG and hepatic carcinoma HepG2 cells in vitro.     

Tissue contents and urinary excretion of taurine after administration of L-cysteine and L-2-oxothiazolidine-4-carboxylate to rats

Tissue contents and urinary excretion of taurine were studied in rats after the administration of L-cysteine and its derivatives. Average taurine content in the liver of rats fed a 25% casein diet for 7 days increased 2-fold 2h after the intraperitoneal administration of 5 mmol of L-cysteine per kg of body weight, whereas that in rats fed a 5% casein diet for 2 days increased only slightly. The difference in the liver taurine contents between these two groups was discussed in relation to cysteine dioxygenase. Taurine contents in the heart, brain and blood did not differ significantly between these two groups or between the control and the group of rats which received L-cysteine. The increase in liver taurine concentrations after L-cysteine administration was much higher than that after L-cystine administration, suggesting a difference in their absorption. The intraperitoneal administration of 5 mmol/kg of L-2-oxothiazolidine-4-carboxylate (OTCA) resulted in a 3-fold increase in liver taurine content. The average increase in taurine excretion in the 24-h urine after OTCA administration corresponded to about 6.0% and that in the next 24-h urine to about 2.6% of OTCA administered, suggesting that nearly 10% of OTCA was metabolized to taurine and excreted in the urine.     

Inhibition of bacterial adhesion of uropathogenic Escherichia coli strains by the urine of patients treated with nitroxoline

The present study evaluates the effects of sub-inhibitory concentrations of nitroxoline, (oxyquinoline derivative) widely used in the treatment of uncomplicated, urinary tract infections, on the adherence of uropathogenic strains of Escherichia coli. These bacterial strains showed mannose sensitive and/or mannose resistant hemagglutinating activity (HA). In the presence of nitroxoline and at sub-MIC concentrations, inhibition of adherence is 90% (MIC/4), 87% (MIC/8), and 70% (MIC/16), whatever HA's are expressed by the E. coli strains. The inhibitory effect on adherence is also observed in the urine after oral administration of 400 mg of nitroxoline. The concentrations of nitroxoline in the urine are determined by microbiological assay (anti-bacterial activity) and by physico-chemical assay (total nitroxoline and free nitroxoline). The percentages of inhibition are related to the concentrations of free and conjugated nitroxoline. For a 1/16 dilution of urine, the inhibitory effect is 70% and 87% respectively 1 h 30 and 2 h 30 after oral administration of nitroxoline. After 5 h, a similar inhibitory effect is observed for a 1/2 dilution of urine. These results justify the performance of a clinical trial on the prophylaxis of recurrent urinary tract infections by nitroxoline.     

Benzyl isothiocyanate sensitizes human pancreatic cancer cells to radiation by inducing apoptosis

Isothiocyanates are a class of naturally occurring chemopreventive agents known to suppress proliferation of cancer cells in culture. The present study was undertaken in order to examine the effects of benzyl isothiocyanate (BITC), one of the common dietary isothiocyanates, on the radiosensitivity of human pancreatic cancer cells and to gain insights into the underlying molecular mechanism of BITC-induced radiosensitization. Two human pancreatic cancer cell lines, PANC-1 and MIAPaCa-2, were treated with BITC and irradiated with X-rays. Radiation sensitivity, apoptosis, and protein levels were determined by a clonogenic assay, fluorescence microscopic analysis with DAPI staining and Western blotting, respectively. MIAPaCa-2 cells were relatively more sensitive to BITC treatment compared with PANC-1 cells. Radiosensitization was observed in both PANC-1 and MIAPaCa-2 cells incubated with BITC at 5 to 10 μM and 2.5 to 5 μM for 24 h, respectively. The combination treatments with BITC and X-rays also revealed an increased percentage of apoptotic cells. In addition, treatment with BITC and X-rays resulted in a decrease in the protein levels of the X-linked inhibitor of apoptosis (XIAP), inhibitor of apoptosis (IAP) family protein, and in a marked increase in the apoptosis protease activating factor-1 (Apaf-1), essential for activation of caspase-9 in stress-induced apoptosis. BITC may be a useful radiosensitizer for radiotherapy of pancreatic cancers.