Urinary excretion of biomarkers for radical-induced damage in rats treated with NDMA or diquat and the effects of calcium carbimide co-administration.

The urinary excretion of seven aldehydes, acetone, coproporphyrin III and 8-hydroxy-2'-deoxyguanosine (8-OH-dG) as non-invasive biomarkers of oxidative damage was measured in rats treated with diquat or N-nitrosodimethylamine (NDMA), two compounds causing hepatic damage by different mechanisms. Furthermore, the effect of co-administration of the aldehyde dehydrogenase inhibitor, calcium carbimide (CC) on the urinary excretion of the aldehydes was determined. Slight hepatotoxicity was found at the end of the experiment after treatment with NDMA (0.5, 4 and 8 mg/kg at t = 0, 48 and 96 h, respectively) or diquat (6.8 and 13.6 mg/kg at t = 0 and 48 h, respectively). In diquat treated rats slight nephrotoxicity was also found. Urinary excretion of aldehydes, acetone and coproporphyrin III remained largely unchanged in rats treated with NDMA. In the rats treated with diquat, the urinary excretion of several aldehydes was several-fold increased. An increase was also found in the urinary excretion of 8-OH-dG after the second dose of diquat. Treatment of rats with CC did not significantly influence the urinary excretion of aldehydes in control and NDMA rats. However, in rats treated with diquat, CC caused a potentiating effect on the excretion of acetaldehyde, hexanal and malondialdehyde (MDA), indicating that oxidation of aldehydes to carbonylic acids by aldehyde dehydrogenases (ALDHs) might be an important route of metabolism of aldehydes. In conclusion, increased urinary excretion of various aldehydes, acetone, coproporphyrin III and 8-OH-dG was observed after administration of diquat, probably reflecting oxidative damage induced by this compound. No such increases were found after NDMA administration, which is consistent with a different toxicity mechanism for NDMA. Therefore, excretion of aldehydes, acetone, coproporphyrin III and 8-OH-dG might be used as easily accessible urinary biomarkers of free radical damage.

Inhibition of various steps in the replication cycle of vesicular stomatitis virus contributes to its photoinactivation by AlPcS4 or Pc4 and red light.

Vesicular stomatitis virus (VSV) was used as a model virus to study the processes involved in photoinactivation by aluminum phthalocyanine tetrasulfonate (AlPcS4) or silicon phthalocyanine HOSiPcOSi(CH3)2(CH2)3N(CH3)2 (Pc4) and red light. Previously a very rapid decrease in the intracellular viral RNA synthesis after photodynamic treatment was observed. This decrease was correlated to different steps in the replication cycle. Binding of VSV to host cells and internalization were only slightly impaired and could be visualized by electron microscopy. The capability of the virus to fuse with membranes in an acidic endosomal environment was studied using both pyrene-labeled liposomes and a hemolysis assay as a model. These tests indicate a rapid decrease of fusion capacity after AlPcS4 treatment, which correlated with the decrease in RNA synthesis. For Pc4 treatment no such correlation was found. The fusion process is the first step in the replication cycle, affected by AlPcS4 treatment, but also in vitro RNA polymerase activity was previously shown to be inhibited. Inactivation of VSV by Pc4 treatment is apparently caused by damage to a variety of viral components. Photodynamic treatment of virus suspensions with both sensitizers causes formation of 8-oxo-7,8-dihydroguanosine in viral RNA as measured by HPLC with electrochemical detection. This damage might be partly responsible for inhibition of the in vitro viral RNA polymerase activity by photodynamic treatment.

Urinary excretion of biomarkers of oxidative kidney damage induced by ferric nitrilotriacetate.

There is an increasing need for biomarkers of oxidative stress in animals and man. In this study, we have evaluated in the rat the utility of various endogenous products that are excreted in urine as potential noninvasive biomarkers of oxidative stress in the kidney. Renal oxidative damage was induced by daily i.p. injections of ferric nitrilotriacetate (Fe-NTA) for a period of 13 days. The daily dose of Fe-NTA was increased during the experiment from 6 to 40 mg Fe/kg body wt. The levels of 8-hydroxy-2'-deoxyguanosine (8-OHdG), coproporphyrin III (COPRO III), seven aldehydes, and acetone were determined in fractionated urine samples and compared with commonly used urinary and plasma clinical chemical parameters for toxicity. The parameters that showed the earliest increase were acetaldehyde (ACET), propanal (PROPA), and COPRO III. Their increase was significantly earlier than that of classical clinical chemical parameters indicative of renal damage such as urinary concentration of glucose (GLU) and protein (PRT), and N-acetyl-beta-D-glucosaminidase (NAG) activity. The excretion of 8-OHdG was increased only after administration of the highest dose of Fe-NTA. Urinary excretion of acetone, form-aldehyde (FOR), butanal (BUTA), pentanal (PENTA) hexanal (HEXA), and malondialdehyde (MDA) was also increased; however, their increase occurred only slightly before or simultaneously with that of the urinary clinical chemical parameters. In conclusion, 8-OHdG, acetone, FOR, BUTA, PENTA, HEXA, and MDA may possibly serve as biomarkers for oxidative kidney damage. COPRO III, ACET, and PROPA might even be used as biomarkers of production of reactive oxygen species at an early stage.

Evaluation of urinary biomarkers for radical-induced liver damage in rats treated with carbon tetrachloride.

Carbon tetrachloride (CCl4) is a model compound for inducing free radical damage in liver. In this study 10 biomarkers in rats treated i.p. with three different single doses of CCl4 (0.25, 0.50, and 1.00 ml/kg body wt) were measured dose and time dependently and compared to evaluate these urinary products as noninvasive biomarkers for radical damage. Eight degradation products of lipid peroxides, namely, formaldehyde, acetaldehyde, acetone, propanal, butanal, pentanal, hexanal, and malondialdehyde (MDA), 8-hydroxy-2'-deoxyguanosine (8-OH-dG) and coproporphyrin III were measured in this study. As general measures of toxicity, several clinical chemical parameters (n = 12) and histopathological damage were determined. A dose-dependent increase in both the clinical parameters and the lipid degradation products was found. Increases in lipid degradation products were statistically significant at doses of 0.5 and 1 ml/kg CCl4. An increase in these products was already found in the first 12 h after exposure. At the lowest dose, 0.25 ml/kg CCl4, acetaldehyde and propanal already showed a statistically significant increase as well. No change in the urinary levels of 8-OH-dG could be found in this study and a decrease in the urinary excretion of coproporphyrin III was found. It is concluded that 8-OH-dG and coproporphyrin III are not useful biomarkers for radical damage induced by CCl4. Lipid degradation products, however, are promising noninvasive biomarkers for in vivo radical damage, although the precise specificity of these biomarkers for damage induced by radicals needs to be further investigated.

Excretion of multiple urinary biomarkers for radical induced damage in rats treated with three different nephrotoxic compounds.

In the present study the urinary excretion of seven aldehydes, acetone and coproporphyrin III as non-invasive in vivo biomarkers of free radical damage was measured in rats after treatment with three nephrotoxic compounds: cisplatin, mercuric chloride (HgCl2) and N -acetyl- S -(1,1,2,2-tetrafluoroethyl)-L-cysteine (TFE-Nac). A clear difference between the different nephrotoxic compounds was found in the time interval between dosage and maximal toxicity, as measured by clinical chemical parameters in urine. In rats treated with TFE-Nac and HgCl2 this was fast: 12 h and 24 h after treatment, respectively. In the rats treated with cisplatin, however, nephrotoxicity occurred later: 96 h-108 h after treatment. Urinary creatinine excretion was decreased in all treatments. Therefore, the excretion of the proposed biomarkers was expressed as amount excreted per 12 h urine fraction as well as amount excreted per mol creatinine in each 12 h urine fraction. Urinary excretion of coproporphyrin III was decreased in almost all 12 h urine fractions with all treatments, however, when expressed per mol creatinine, increases were found in urine of rats treated with cisplatin and HgCl2. In cisplatin-treated rats an increase was found in the excretion of formaldehyde per 12 h, but acetaldehyde, propanal and MDA levels were decreased. Expressed per mol creatinine, MDA levels were decreased, but other aldehydes were increased. In HgCl2-treated rats urinary aldehyde excretion expressed per mol creatinine was increased. In TFE-Nac treated animals the urinary levels of acetaldehyde per 12 h were increased and per mol creatinine the levels of some aldehydes were only slightly increased. With none of the treatments did the increase in the biomarkers expressed per mol creatinine exceed the decrease in creatinine excretion. Similar time intervals were found between dosage and maximal excretion of biomarkers as for the time intervals between dosage and maximal toxicity. With all treatments significant increases in the excretion of acetone were found both per 12 h and per mol creatinine, probably related to the increased glucose excretion. It was concluded that no convincing evidence for free radical damage was found in the present study with the employed biomarkers.

Simultaneous determination of eight lipid peroxidation degradation products in urine of rats treated with carbon tetrachloride using gas chromatography with electron-capture detection.

One of the major processes that occur as a result of radical-induced oxidative stress is lipid peroxidation (LPO). Degradation of lipid peroxides results in various products, including a variety of carbonyl compounds. In the present study eight different lipid degradation products, i.e., formaldehyde, acetaldehyde, acetone, propanal, butanal, pentanal, hexanal and malondialdehyde were identified and measured simultaneously and quantitatively in rat urine after derivatization with O-(2,3,4,5,6-pentafluorbenzyl)hydroxylamine hydrochloride, extraction with heptane and using gas chromatography-electron-capture detection (GC-ECD). The identity of the respective oximes in urine was confirmed by gas chromatography-negative ion chemical ionization mass spectrometry (GC-NCI-MS). Simultaneously measured standard curves were linear for all oxime-products and the detection limits were between 39.0 +/- 5.3 (n=9) and 500 +/- 23 (n=9) fmol per microl injected sample. Recoveries of all products from urine or water were 73.0 +/- 5.2% and higher. In urine of CCl4-treated rats an increase in all eight lipid degradation products in urine was found 24 h following exposure. ACON showed the most distinct increase, followed by PROPA, BUTA and MDA. It is concluded that the rapid, selective and sensitive analytical method based on GC-ECD presented here is well suited for routine measurement of eight different lipid degradation products. These products appear to be useful as non-invasive biomarkers for in vivo oxidative stress induced in rats by CCl4.

Disposition in rat of [2-3H]-trans-4-hydroxy-2,3-nonenal, a product of lipid peroxidation.

1. 4-Hydroxy-2,3-nonenal (HNE) is an end product of lipid peroxidation (LPO) and a well known cytotoxic aldehyde that exhibits a variety of biological effects. In this study the in vivo disposition and covalent binding of i.p. administered [2-3H]HNE was examined in the rat. 2. It was found that several metabolites of [2-3H]HNE are excreted in urine among which at least four mercapturic acids. 1,4-Dihydroxynonane mercapturic acid (DHN-MA) appeared to be the most abundant mercapturic acid excreted in urine (3.5% of the dose) and the excretion of the other three mercapturic acids amounted to 2% of the dose. 3. Within 48 h following i.p. administration of 5 or 25 mumol/kg bodyweight [2-3H]HNE (specific activity 4 microCi/mumol) about 25% of the radioactivity was excreted in urine, whereas 18% of the radioactivity appeared in the faeces. 4. After 48 h, 7% of the radioactivity was still present in the liver and 0.2% in other organs, but this radioactivity appeared to not to be covalently bound to cellular macromolecules. It was found that only 0.13% of the radioactivity was covalently bound in the liver and even less in other organs.