ROS/JNK-mediated lysosomal injury in rat intestinal epithelial-6 cells during heat stress.

Injury to the intestinal epithelial cells and loss of the intestinal barrier are critical to heatstroke. To reveal the mechanism through which heatstroke leads to intestinal epithelial injury, the relationship between reactive oxygen species (ROS), c-Jun NH2-terminal kinase (JNK), and lysosomes were studied in intestinal epithelial cells subjected to heat stress. Cells of heat stress groups were incubated at 43 °C for 1 h, then incubated at 37 °C as indicated. Control group cells were incubated at 37 °C. Cell-counting kit-8 assay was used to assess cell viability. Cells were labeled with 2'-7'dichlorofluorescin diacetate and acridine orange (AO) staining, respectively, the total ROS and AO were detected by confocal laser scanning microscopy and flow cytometry. Apoptosis was analyzed by flow cytometry using annexin V-fluorescein isothiocyanate/prodium iodide staining, the expressions of mitogen-activated protein kinases were detected by western blotting. Heat stress induced apoptosis and inhibited cell viability, the production of ROS, and lysosomal injury in IEC-6 cells. After pretreatment with the lysosomal cathepsin inhibitor E64, the JNK inhibitor SP600125, or the ROS scavenger NAC, the effect of heat stress on apoptosis or lysosomal injury was significantly attenuated. In conclusion, heat stress induced apoptosis, lysosomal injury, and the accumulation of ROS in IEC-6 cells; mechanistically, this occurred through the ROS-induced activation of JNK signaling, which mediated the lysosomal injury and ultimately apoptosis.

2,3,6-triaminopyridine, a metabolite of the urinary tract analgesic phenazopyridine, causes muscle necrosis and renal damage in rats.

Some aromatic polyamines form very stable free radicals and readily undergo autoxidation with concomitant formation of 'active oxygen' species. These substances cause necrosis of striated muscle in rats, and it has been suggested that this is due to free radical formation and disruption of energy production through their oxidation via the cytochrome c/cytochrome oxidase system of mitochondria. 2,3,6-Triaminopyridine, which is structurally related to the myotoxic amines and likewise undergoes autoxidation and disrupts mitochondrial metabolism, is a metabolite of the widely used urinary analgesic phenazopyridine. When administered to rats, triaminopyridine caused extensive necrosis of skeletal muscle and a lesser degree of damage to heart muscle. It also induced vacuolation and necrosis of distal tubules of the kidney, associated with tubular dilatation and cast formation. Both muscle damage and renal tubular necrosis have been reported following use or abuse of phenazopyridine, and it is likely that triaminopyridine is responsible for both of these effects.

Urinary excretion of pyridinium cross-links of collagen in infancy.

This cross-sectional study evaluated urinary excretion of pyridinium cross-links of collagen, specific markers of ongoing bone resorption, in infants aged 1 week to 7 months and examined the relationship between urinary cross-links and individual renal function. Spot urines from a total of 100 infants were analyzed. The collagen cross-links, pyridinoline (Pyd) and deoxypyridinoline (D-Pyd), were assayed by fluorescence detection after high-performance liquid chromatography (HPLC). Beta2-Microglobulin (beta2M), an index of renal tubular function, was determined by radioimmunoassay. In healthy term infants, urinary collagen cross-links were several times higher than the reported data for older children, with peak values seen at 1 month of age. Excretion of Pyd and D-Pyd was also markedly elevated in 1-month-old preterm infants, despite poor somatic growth. Such high excretion of collagen cross-links probably reflects the state of accelerated bone turnover in infancy. The postnatal change in the cross-links was different from that in urinary beta2M, and the values obtained did not correlate with beta2M in either term or preterm infants. These results indicate that cross-link excretion is not influenced directly by individual renal function.

Generation of superoxide radical and hydrogen peroxide by 2,3,6-triaminopyridine, a metabolite of the urinary tract analgesic phenazopyridine.

2,3,6-Triaminopyridine, a metabolite of the widely-used drug phenazopyridine, has been shown to autoxidize at neutral pH, generating superoxide radical and hydrogen peroxide. Hydrogen peroxide was also detected in erythrocytes exposed to this substance, and these cells suffered oxidative damage, as reflected by methaemoglobin formation and glutathione depletion. The in vitro effects of 2,3,6-triaminopyridine are closely similar to those of the structurally-related compound, 1,2,4-triaminobenzene. The latter substance is known to be highly toxic in vivo by mechanisms which may involve free radical production and oxidative stress. It is possible, therefore, that triaminopyridine may be similarly toxic in animals and that this metabolite could be responsible for some of the harmful side-effects associated with phenazopyridine use.

Metabolism and disposition of phenazopyridine in rat.

1. The blood profile, tissue distribution, biliary and urinary excretion, and metabolism of 14C-phenazopyridine (PAP) was studied in male Wistar rats. 2. Based on the blood profile of 14C the absorption of PAP from the gastrointestinal tract was rapid; the t1/2 of elimination was 7.35 h. 3. Biliary excretion was a major route of elimination with 40.7% dose excreted by this route in bile duct-cannulated rats over the 0-8 h period. The predominant metabolite was conjugated 4'-hydroxy-PAP. 4. Liver and kidney showed the highest tissue levels of PAP-derived 14C, and significant covalent binding was found in these two tissues. 5. The major urinary metabolite of PAP was 4-acetylaminophenol (NAPA) followed in order by 5,4'-dihydroxy-PAP, 5-hydroxy-PAP, 4'-hydroxy-PAP and 2'-hydroxy-PAP; unchanged PAP accounted for < 1% dose. 6. Doubling the dose of PAP to 200 mg/kg caused a proportionate decrease in urinary NAPA excretion and an increase in 5-hydroxy-PAP.

Mutagenicity of structurally related phenylazo-3-pyridines in Salmonella typhimurium.

Studies were conducted to explore structure-activity relationships for 4'-N,N-dimethylamino-1'-phenylazo-3-pyridine and nine structurally related compounds in Salmonella typhimurium tester strains TA1535, TA100, TA1537, TA1538, TA98. Each compound was tested for mutagenicity at five or more concentrations that varied from 10-5000 micrograms/plate. We used the standard plate test and the investigations were carried out both in the absence and presence of Aroclor-1254-induced rat-liver homogenate and the components of the NADPH-generating system. Negative response was observed for 4'-N,N-dimethylamino-1'-phenylazo-3-pyridine and five of its analogues (4'-N,N-diethylamino-1' phenylazo-3-pyridine; 4'-N,N-di-(beta-hydroxyethylamino)-1' phenylazo-3-pyridine; 4'-N-methylamino sulfonic acid-1'-phenylazo-3-pyridine; 4'-N,N-dimethylamino-6'-acetamido-1' phenylazo-3-pyridine, and 4'-N,N-di-(beta-hydroxyethylamino)-6'-methyl-l' phenylazo-3-pyridine). When S9 induced by Aroclor-1254 was present, the compound 4'-N,N-dimethylamino-6-methoxy-1' phenylazo-3-pyridine exhibited mutagenic activity in the two strains TA1538 and TA98. The compound 4',6'-diamino-3-methyl-1'-phenylazo-3-pyridine was also mutagenic, both in the presence and in the absence of S9 mix. The two compounds 4'-N,N-dimethylamino-6-butoxy-1'-phenylazo-3-pyridine and 4'N,N-di-(beta-hydroxyethylamino)-1'-phenylazo-3-[6-N,N-di-(beta- hydroxyethylamino) pyridine were either weakly mutagenic or nonmutagenic. On the basis of these data, it is concluded that the mutagenicity of phenylazo-3-pyridines, like monocyclic aromatic amines and azo dyes, is influenced by the nature of the substituent chemical groups and their positions in the molecular structure of the compounds.

Effect of some additives on the properties of phenazopyridine hydrochloride granules and their corresponding tablets.

The effect of the diluents, lactose and calcium carbonate and of the binders, syrup, gelatin, methylcellulose and Eudragit E on the physical properties of phenazopyridine hydrochloride (PNHCl) granules was evaluated. A correlation existed between the granules' physical properties and those of their compressed tablets. With regard to drug release, lactose-syrup 30% was the best of all diluent-binder combinations, followed by lactose-methylcellulose 4%. Also lactose was found to be superior to calcium carbonate in drug release when gelatin and methylcellulose were used as binders. Eudragit E was the best binder with calcium carbonate in this respect. On the other hand, the bioavailability of PNHCl in humans was the same when lactose was used with either gelatin, syrup or methylcellulose, but higher than that obtained with a combination of calcium carbonate and Eudragit E 15%.

Nephrotoxicity of phenazopyridine.

A young man developed reversible acute renal failure after a large overdose of phenazopyridine. The renal failure was treated by peritoneal dialysis. Analysis of blood and urine samples failed to demonstrate the parent drug but a metabolite with similar ultraviolet absorption was detected. No parent drug or metabolites were detected in the peritoneal dialysate.

Acquired methemoglobinemia and hemolytic anemia after usual doses of phenazopyridine.

Two patients developed symptomatic methemoglobinemia and hemolytic anemia after treatment with phenazopyridine. Methemoglobinemia appears to be a rare occurrence after commonly used doses of phenazopyridine; phenazopyridine-associated hemolytic anemia has been reported both after overdose and after usual doses. The presentation of methemoglobinemia in the first patient and the response to treatment with methylene blue in the second patient were unusual, suggesting that the patients had a red cell defect or were exposed to other oxidizing substances. One of the major metabolites of phenazopyridine is aniline, a known cause of methemoglobinemia. Aniline-induced methemoglobinemia is less responsive to treatment with methylene blue than nitrate- or nitrite-induced methemoglobinemia. This may explain, in part, the poor response to methylene blue by one of our patients.