JP-8 jet fuel-induced DNA damage in H4IIE rat hepatoma cells

We investigated the genotoxicity of middle distillate jet fuel, Jet Propulsion 8 (JP-8), on H4IIE rat hepatoma cells in vitro. DNA damage was evaluated using the comet (single cell gel electrophoresis) assay. Cells were exposed for 4h to JP-8 (solubilized in ethanol (EtOH) at 0.1% (v/v)) to concentrations ranging from 1 to 20microg/ml. Exposure to JP-8 resulted in an overall increase in mean comet tail moments ranging from 0.74+/-0.065 (0.1% EtOH control) to 3.13+/-0.018,4.36+/-0.32,5.40+/-0.29,7.70+/-0.52 and 11.23+/-0.77 for JP-8 concentrations 3, 5, 10, 15 and 20microg/ml, respectively. Addition of DNA repair inhibitors hydroxyurea (HU) and cytosine arabinoside (Ara-C) to cell culture with JP-8 resulted in accumulation of DNA damage strand breaks and increase in comet tail length. Inclusion of 4mM HU and 40microM Ara-C with 3, 5, 10 and 20microg/ml JP-8 concentrations resulted in increased mean tail moments to 5.94+/-0.43,10.12+/-0.72,17.03+/-0.96,and29.25+/-1.55. JP-8, in the concentrations used in this study, did not result in cytotoxicity or significant apoptosis, as measured using the terminal deoxynucleotidyl transferase (TDT)-mediated dUTP-X nick end labeling (TUNEL) assay. These results demonstrate that relevant exposures to JP-8 result in DNA damage to H4IIE cells, and suggest that DNA repair is involved in mitigating these effects.     

Dental amalgam mercury exposure in rats

The aim of this study was to measure the distribution of mercury, in tissues of rats exposed to amalgam over a two months period. Possible interaction of mercury with copper and zinc in organs was also evaluated. Rats were either exposed to mercury from 4 dental amalgams, or fed the diet containing powdered amalgam during two months. Mercury was measured in the kidney, liver and brain, copper in kidney and brain and zinc in kidney. The results showed significantly higher concentrations of mercury in the kidneys and the brains of rats in both exposed groups compared to control. Even after two months of exposure to mercury brain mercury concentration in rats with amalgam fillings was 8 times higher than in the control and 2 times higher than in rats exposed to amalgam supplemented diet. The highest mercury concentration in the latter group was found in the kidneys and it was 5 times higher than in the control group. We found no significant differences between mercury levels in exposed and control rat's liver. Exposure to mercury from dental amalgams did not alter the concentrations of copper and zinc in the tissues. Histopathological analyses of rats tissues did not show any pathological changes. These results support previously proposed nose-brain transport of mercury released from dental amalgam fillings.     

Biological availability of selenosugars in rats

The biological availability and metabolism of two selenosugars orally administered to rats were investigated. Two other selenium species, selenite and trimethylselenonium ion (TMSe) were included in the study as positive and negative controls, respectively. Male Wistar strain rats (three per group) at 8 weeks of age were exposed to sodium selenite, TMSe, selenosugar 1 (methyl-2-acetamido-2-deoxy-1-seleno-beta-D-galactopyranoside) or selenosugar 2 (methyl-2-acetamido-2-deoxy-1-seleno-beta-D-glucopyranoside) through drinking water for 48 h. Total selenium concentrations (ICPMS) and selenium species concentrations (HPLC/ICPMS) were determined in urine samples collected in two 24h periods during the exposure, and total selenium concentrations in liver, kidney, small intestine and blood were determined at the end of the experiment. The major species found in background urine were selenosugar 1 (major metabolite) and TMSe (minor metabolite). Rats exposed to selenite excreted large quantities of selenosugars and TMSe consistent with efficient uptake and biotransformation of selenite, whereas TMSe-exposed rats excreted large quantities of TMSe, but there was no significant increase of other selenium metabolites, consistent with TMSe being taken up and excreted unchanged. Rats exposed to selenosugars, however, excreted significant quantities of TMSe suggesting that the sugars were at least partly biologically available and biotransformed. Rats exposed to selenite accumulated selenium in the liver, kidney, small intestine and blood, whereas no accumulation was observed for the other samples except for small increases in selenium concentrations of small intestine from the two selenosugar-exposed groups.     

Effects of whole body microwave exposure on the rat brain contents of biogenic amines.

The effects of whole body microwave exposure on the central nervous system (CNS) of the rat were investigated. Rats weighing from 250 to 320 g were exposed for 1 h to whole body microwave with a frequency of 2450 MHz at power densities of 5 and 10 mW.cm-2 at an ambient temperature of 21-23 degrees C. The rectal temperatures of the rats were measured just before and after microwave exposure and mono-amines and their metabolites in various discrete brain regions were determined after microwave exposure. Microwave exposure at power densities of 5 and 10 mW.cm-2 increased the mean rectal temperature by 2.3 degrees C and 3.4 degrees C, respectively. The noradrenaline content in the hypothalamus was significantly reduced after microwave exposure at a power density of 10 mW.cm-2. There were no differences in the dopamine (DA) content of any region of the brain between microwave exposed rats and control rats. The dihydroxyphenyl acetic acid (DOPAC) content, the main metabolite of DA, was significantly increased in the pons plus medulla oblongata only at a power density of 10 mW.cm-2. The DA turnover rates, the DOPAC:DA ratio, in the striatum and cerebral cortex were significantly increased only at a power density of 10 mW.cm-2. The serotonin (5-hydroxytryptamine, 5-HT) content in all regions of the brain of microwave exposed rats was not different from that of the control rats. The 5-hydroxyindoleacetic acid (5-HIAA) content in the cerebral cortex of microwave exposed rats was significantly increased at power densities of 5 and 10 mW.cm-2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Estrogen induction of ornithine aminotransferase in rat kidney slices

Immunologic titration of kidney extracts from control and estradiol-injected rats with anti-ornithine aminotransferase serum shows that the hormone induces the enzyme by increasing its concentration in the kidney. The induction has been studied in vitro with kidney slices. Several-fold increase in the rate of ¹⁴C-leucine incorporation into the enzyme by hexestrol has been demonstrated. In addition, certain antiestrogens also induce the enzyme synthesis in vitro. These results show that the induction of this enzyme in the kidney does not require the participation of an estrogen metabolite from another tissue.     

Effects of nickel poisoning on expression pattern of the 72/73 and 94 kDa stress proteins in rat organs and in the COS-7, HepG2, and A549 cell lines

The present study deals with the effects of Ni on the expression level of three stress proteins, namely, the cytosolic HSP72 and HSP73, and the reticulum-associated GRP94. Experiments were carried out on "Wistar' female rats daily injected with 4 mg NiCl2 per kg body weight for 1, 3, 5, and 10 days. Another set of experiments were carried out using cell lines, derived from the monkey kidney (COS-7), and from human tumors of the lung (A549) and liver (HepG2). Cells were cultured for 4 days in the permanent presence of 100, 200, or 400 microM NiCl2. In control rats, stress proteins pattern was found to be tissue specific: two protein bands of 96 and 94 kDa were immunodetected with the anti-GRP94 antibody in kidney and liver extracts, whereas only the 96 kDa band was present in ovary extracts. HSP73 was present in kidney, liver, and ovary whereas HSP72 was only found in kidney. In kidney of nickel-treated animals, HSP73 and the 96 kDa proteins were overexpressed whereas HSP72 was strongly down regulated. No such effect was observed in liver or ovary. Similarly, in nickel-treated cell lines, HSP72 was downregulated and GRP94 (96 kDa protein) was overexpressed. HSP73 expression appeared moderately increased in A549 cells but decreased in COS-7 cells. Because long-term caloric restriction was reported to reduce free radical generation in cells, the effect of 1 month food restriction (50%) was tested in rats as a possible way to lower oxidative damages induced by Ni. No significant effect on HSP expression was observed.     

Analysis of rat testicular protein expression following 91-day exposure to JP-8 jet fuel vapor

We analyzed protein expression in preparations from whole testis in adult male Sprague-Dawley rats exposed for 6 h/d for 91 consecutive days to jet propulsion fuel-8 (JP-8) in the vapor phase (0, 250, 500, or 1000 mg/m(3) +/- 10%), simulating a range of possible human occupational exposures. Whole body inhalation exposures were carefully controlled to eliminate aerosol phase, and subjects were sacrificed within 48 h postexposure. Organ fractions were solubilized and separated via large-scale, high resolution two-dimensional electrophoresis, and gel patterns scanned, digitized and processed for statistical analysis. Seventy-six different testis proteins were significantly increased or decreased in abundance in vapor-exposed groups, compared to controls, and dose-response profiles were often nonlinear. A number of the proteins were identified by peptide mass fingerprinting and related to histopathological or physiological deficits shown in previously published studies to occur with repeated exposure to hydrocarbon fuels or solvents. These results demonstrate a significant effect of JP-8 exposure on protein expression, particularly in protein expression in the rodent testis, and suggest that a 91 d exposure to jet fuel vapor induces changes of equal or greater magnitude to those reported previously for shorter duration JP-8 aerosol exposures.     

Metabolic regulatory network alterations in response to acute cold stress and ginsenoside intervention

Acute stress may trigger systemic biochemical and physiological changes in living organisms, leading to a rapid loss of homeostasis, which can be gradually reinstated by self-regulatory mechanisms and/or drug intervention strategy. However, such a sophisticated metabolic regulatory process has so far been poorly understood, especially from a holistic view. Urinary metabolite profiling of Sprague-Dawley rats exposed to cold temperature (-10 degrees C) for 2 h using GC/MS in conjunction with modern multivariate statistical techniques revealed drastic biochemical changes as evidenced by fluctuations of urinary metabolites and demonstrated the protective effect of total ginsenosides (TGs) in ginseng extracts on stressed rats. The metabonomics approach enables us to visualize significant alterations in metabolite expression patterns as a result of stress-induced metabolic responses and post-stress compensation, and drug intervention. Several major metabolic pathways including catecholamines, glucocorticoids, the tricarboxylic acid (TCA) cycle, tryptophan (nicotinate), and gut microbiota metabolites were identified to be involved in metabolic regulation and compensation required to restore homeostasis.     

Liver, lung and kidney homogenates used as an activation system in mutagenicity studies of airborne particles and of expectorate and urine samples from exposed workers in a coke plant

A comparison was made between lung and kidney homogenates on the one hand and liver S9 from rats on the other hand in order to compare their ability to activate promutagens. The Salmonella reversion assay was used on extracts of airborne particles from the top of coke oven batteries, and of expectorate and urine samples from exposed workers in the same coke plant. The contents of benzo[a]anthracene and benzo[a]pyrene in the different test solutions were measured by high-resolution gas chromatography/mass spectrometry. Both mutagens were detected in the filter extract and in the expectorates from the exposed workers but not in the expectorates from the control groups or in the urine samples. The liver S9 gave significantly higher mutagenicity than lung and kidney activation with both filter samples and expectorate and urine samples.     

Stress induced in heart and other tissues by rat dermal exposure to JP-8 fuel

Limited information is available regarding the development of systemic organ stress by dermal exposure to JP-8 fuel. In this study, the systemic stress potential of this fuel is evaluated in a rat model subjected to dermal applications of JP-8 for 7 days at 300 μl per day. Tissue histology indicated that JP-8 induces morphological alterations that suggest that tissue stress in the heart is more substantial than stress in the kidney and liver. Immunoblot analysis of tissues revealed increased levels of the inducible heat shock protein 70 (HSP70) in the heart, kidney, and liver after this dermal JP-8 exposure. This exposure also leads to increased levels of heme oxygenase-1 (HO-1/HSP3) in the liver. Additionally during this exposure, a negative regulator of inflammation, IκBα (inhibitor of NF-κB), was increased in the liver, slightly increased in the kidney, and not increased in the heart. Two regions of the rat brain were also examined and HSP70 and IκBα were increased in the cerebellum but not significantly increased in the cortex. This study indicates dermal JP-8 exposure causes systemic alterations that are associated with cytoprotective activities (e.g., in the liver) as well as potentially toxic mechanisms (heart and kidney).     

A relationship between hepatic metabolism of reduced lantadene A and its toxicity in rats and sheep

The metabolism of the cholestatic triterpene acid reduced lantadene A has been studied in susceptible and resistant rats and in sheep which are susceptible to intoxication. Sheep and susceptible female rats produced a similar major metabolite and rats produced a second metabolite which was a glucuronide. These metabolites were also observed in extracts of bile canalicular membranes prepared from intoxicated rats. Resistant male and female rats produced a similar major metabolite which was different to those synthesized by susceptible animals. It is concluded that in rats and sheep there is a correlation between the type of metabolites produced in the liver and the susceptibility to intoxication by reduced lantadene A.     

23-carboxy-24,25,26,27-tetranorvitamin D3 (calcioic acid) and 24-carboxy-25,26,27-trinorvitamin D3 (cholacalcioic acid): end products of 25-hydroxyvitamin D3 metabolism in rat kidney through C-24 oxidation pathway

During the past two and half decades the elucidation of the metabolic pathways of 25OHD(3) and its active metabolite 1alpha,25(OH)(2)D(3) progressed in parallel. In spite of many advances in this area of vitamin D research, the unequivocal identification of the end products of 25OHD(3) metabolism through C-24 oxidation pathway has not been achieved. It is now well established that both 25OHD(3) and 1alpha,25(OH)(2)D(3) are metabolized through the same C-24 oxidation pathway initiated by the enzyme 24-hydroxylase (CYP24A1). Based on the information that the end product of 1alpha,25(OH)(2)D(3) metabolism through C-24 oxidation pathway is 1alpha-OH-23- COOH-24,25,26,27-tetranor D(3) or calcitroic acid; the metabolism of 25OHD(3) into 23-COOH-24,25,26,27-tetranor D(3) has been assumed. Furthermore, a previous study indicated 24-COOH-25,26,27-trinor D(3) as a water soluble metabolite of 24R,25(OH)(2)D(3) produced in rat kidney homogenates. Therefore, 24-COOH-25,26,27-trinor D(3) was also assumed as another end product of 25OHD(3) metabolism through C-24 oxidation pathway. We embarked on our present study to provide unequivocal proof for these assumptions. We first studied the metabolism of 25OHD(3) at low substrate concentration (3x10(-10)M) using [1,2-(3)H]25OHD(3) as the substrate in the perfused rat kidneys isolated from both normal and vitamin D(3) intoxicated rats. A highly polar water soluble metabolite, labeled as metabolite X was isolated from the kidney perfusate. The amount of metabolite X produced in the kidney of a vitamin D intoxicated rat was about seven times higher than that produced in the kidney of a normal rat. We then produced metabolite X in a quantity sufficient for its structure identification by perfusing kidneys isolated from vitamin D intoxicated rats with high substrate concentration of 25OHD(3) (5x10(-6)M). Using the techniques of electron impact and thermospray mass spectrometry, we established that the metabolite X contained both 23-COOH-24,25,26,27-tetranor D(3) and 24-COOH-25,26,27-trinor D(3) in a ratio of 4:1. The same metabolite X containing both acids in the same ratio of 4:1 was also produced when 24R,25(OH)(2)D(3) was used as the starting substrate. Previously, the trivial name of cholacalcioic acid was assigned to 24-COOH-25,26,27-trinorvitamin D(3). Using the same guidelines, we now assign the trivial name of calcioic acid to 23-COOH-24,25,26,27-tetranor D(3). In summary, for the first time our study provides unequivocal evidence to indicate that both calcioic and cholacalcioic acids as the end products of 25OHD(3) metabolism in rat kidney through C-24 oxidation pathway.     

In vitro formation of organ specific proximate carcinogen of benzo(a)pyrene by rat homogenates

In order to determine the organ specific carcinogenicity of benzo(a)pyrene (B(a)P), its metabolites, formed in vitro by incubation with the homogenates from liver, lungs, kidneys, intestine and brain of rats, were isolated by TLC and spectroscopy. B(a)P was found to be converted into a number of metabolites by different tissue homogenates. The results showed that the proximate carcinogenic metabolite, 7,8-dihydro-7,8-dihydroxy B(a)P was formed only when rat lung and kidney homogenates were incubated with B(a)P in vitro. The UV spectral analysis also confirmed the formation of this metabolite only on incubation of B(a)P with rat lung and kidney homogenates. As the proximate carcinogenic metabolite was only formed by incubating B(a)P with the homogenates from target organs, its organ specific carcinogenicity may be explained.     

Enzymatic metabolism of 3-deoxyglucosone,a Maillard intermediate

Summary In order to verify the formation of endogenous 3-deoxyglucosone (3-DG), an intermediate compound in the Maillard reaction, we tried to detect 3-deoxyfructose (3-DF) which is main metabolite of 3-DG. Endogenous 3-DF was detected in the urine of normal and diabetic rats by the oral administration of 3-DG-free feed. Metabolizing activities of crude extracts prepared from porcine organs were examined using methylglyoxal (MG) and 3-DG as substrates. NAD- or NADP-dependent 2-oxoaldehyde dehydrogenase activity was detected in liver, kidney, small intestine and lung. On the other hand, NADH- or NADPH-dependent 2-oxoaldehyde reductase activity was detected in all porcine organs in which liver and kidney contained higher activity of NADPH-dependent enzyme than the other organs. The reductase which catalyzes the reduction of 3-DG to 3-DF and MG to acetol, was purified and characterized from porcine kidney. The enzyme was the same to NADPH-dependent-2-oxoaldehyde reductase from porcine liver, which is speculated to prevent the advanced stage of the Maillard reaction as a self-defense enzyme.     

Reduced high affinity 5 -dihydrotestosterone receptor capacity in the ventral prostate of the aging rat

Cytoplasmic extracts of ventral prostates obtained from young (50–90 day old) rats 24 hours post orchidectomy contained high affinity binding activity for 5α-dihydrotestosterone which migrated with a sedimentation coefficient of 10–11S on linear sucrose gradients. By contrast, when cytoplasmic extracts were prepared from ventral prostates of aging rats (10–14 months old), 80 percent of the animals evidenced a complete absence of 10–11S binding activity. A single extract prepared from the ventral prostates of the remaining aging animals in the 10–14 month age category had a level of 10–11S binding activity that was about 20 to 25 percent of the value routinely obtained for identical preparations from young animals. Cytoplasmic extracts prepared from the ventral prostate of animals older than 14 months were always found to be devoid of 10–11S binding activity.     

Different responses of atrial natriuretic peptide secretion and its receptor density to salt intake in rats

This study investigated whether high-salt intake influences atrial natriuretic peptide (ANP) system, atrial content, and release rate of ANP as well as receptor density in the kidney were measured in salt intake rats. Male Sprague-Dawley rats received either 0.9% or 2% salt in their drinking water for 10 days. The stretch-induced ANP secretion from isolated perfused non-beating left atria was accentuated, and the production of cGMP by ANP in renal cortical tissue membranes were pronounced in rats exposed to 0.9% salt for 10 days but not in rats exposed to 2% salt. The levels of ANP receptor density and expression in renal cortex were decreased in 2% salt intake rats but not in 0.9% salt intake rats. No significant differences in atrial and plasma concentrations of ANP and water balance were observed in both salt intakes. Therefore, these results suggest that atrial ANP secretion and its binding sites in the kidney may respond differently to ingested salt concentrations in rats.     

Methyl 5(6)-(alpha-hydroxyphenyl methyl) benzimidazole-2-carbamate and cysticercosis: chemotherapeutic and electron microscopic studies

Methyl 5(6)-(alpha-hydroxyphenyl methyl) benzimidazole-2-carbamate, a major metabolite of mebendazole was evaluated against Cysticercus fasciolaria (larval form of Taenia taeniaeformis) in rats. The metabolite was assessed in various doses. A regimen of 50 mg/kg x 10 (ip), given one day apart, was found to be most effective and killed all the mature cysticerci. On developing cysts, the treatment was initiated in two schedules; 5 days prior to (d-5 to d-1) and 5 days after (d + 6 to d + 10) administration of T. taeniaeformis eggs to rats. The later protocol with 100 mg/kg x 5 dose (ip) resulted in 95% inhibition in the establishment of cysticerci. Activity of mebendazole against mature cysts was parallel to metabolite whereas against developing cysts, it was inferior. The time related topographical changes that occurred in mature C. fasciolaris after treatment with metabolite (50 mg/kg x 10, ip, one day apart) were observed by scanning electron microscopy. There was loss of contractivity, gradual disappearance of microtriches and progressive degeneration of tegument. Similar changes were noticed with mebendazole. The possession of better efficacy and higher safety range [Indian J Exp. Biol, 25 (1987) 871], suggests that the metabolite can be a potential anthelmintic for man and animals.     

Multiple species of ornithine decarboxylase in rat tissues: Effects of dexamethasone

Several species of ornithine decarboxylase were separated by chromatography of rat thymus and kidney extracts on DEAE-Sepharose CL-6B. One major and one minor species were absent from thymus of rats two hours after hormone treatment but otherwise, the elution profile was identical to thymus from control animals. The elution patterns of ODC activity in kidneys of rats treated 2.5 or 5 hours before sacrifice with dexamethasone differ from that of control kidney and from each other. Enzyme from kidneys early after hormone treatment is eluted earlier than enzyme from control tissue, while at 5 hours, the enzyme is eluted much later than in the control. This suggests that the hormone-induced activity is subsequently modified.     

Ring hydroxylation of 25-hydroxycholecalciferol by rat renal microsomes

Two metabolites have been isolated from rat renal microsomes incubated with 25-hydroxycholecalciferol. Postmitochondrial supernatant fractions from kidneys of thyroidectomized and parathyroidectomized rats were incubated with magnesium acetate, potassium acetate, an NADPH generating system, and 25-hydroxycholecalciferol at a level of 20 micrograms/ml postmitochondrial supernatant for 60 min at 30 degrees C. Lipid extracts of the incubation mixtures were purified by silica gel TLC and HPLC. Two peaks were obtained. Metabolite chi 2 eluted at 18 min and metabolite chi 1 at 23 min when chromatographed on a silica column developed with hexane-isopropanol. Metabolites chi 1 and chi 2 were found to have maximal absorbance at 265 nm. Both metabolites were periodate sensitive, indicating vicinal hydroxyl groups. Mass spectral analysis of metabolite chi 2, which was isolated in greater quantity than metabolite chi 1, indicates that metabolite chi 2 had resulted from hydroxylation of the A ring. Results indicate that 25-hydroxycholecalciferol is hydroxylated on carbon 2 or carbon 4 by renal microsomes. Metabolites chi 1 and chi 2, because of similarity in chromatographic migration and periodate sensitivity, are, perhaps, isomers or 2- and 4-hydroxylated metabolites.