Nrf2 and c-Jun regulation of antioxidant response element (ARE)-mediated expression and induction of gamma-glutamylcysteine synthetase heavy subunit gene

gamma-Glutamylcysteine synthetase (gamma-GCS) is a rate-limiting enzyme in the de novo synthesis of glutathione, a known scavenger of electrophiles and reactive oxygen species (ROS). The gamma-GCS gene is expressed ubiquitously and induced coordinately with NAD(P)H:quinone oxidoreductase(1) (NQO1) and glutathione S-transferase Ya (GST Ya) in response to xenobiotics and antioxidants. The antioxidant response element (ARE) is required for expression and induction of these genes. In the current report, we demonstrated that ARE-mediated gamma-GCS gene expression and induction is regulated by similar Nrf and Jun factors as reported earlier for the NQO1 and GST Ya genes. The gamma-GCS gene ARE competed with the binding of nuclear proteins (Nrf + Jun) to the NQO1 gene ARE (hARE). In addition, the overexpression of Nrf2 and Nrf1 with c-Jun significantly up-regulated gamma-GCS ARE-mediated basal expression and beta-naphthoflavone induction of the chloramphenicol acetyltransferase gene in transfected HepG2 cells. Interestingly, Nrf2 + c-Jun was more effective than Nrf1 + c-Jun in the regulation of ARE-mediated gamma-GCS gene expression. Further experiments demonstrated that the c-Jun level within the cells is an important determinant of the level of ARE-mediated gamma-GCS gene expression. Therefore, at higher concentrations of c-Jun, gamma-GCS gene expression is repressed, presumably due to generation of a sufficient amount of c-Jun + c-Fos complex that interferes with the binding of Nrf2 + c-Jun complex to the ARE.     

Cardiac and pulmonary oxidative stress in rats exposed to realistic emissions of source aerosols

In vivo chemiluminescence (CL) is a measure of reactive oxygen species in tissues. CL was used to assess pulmonary and cardiac responses to inhaled aerosols derived from aged emissions of three coal-fired power plants in the USA. Sprague-Dawley rats were exposed to either filtered air or: (1) primary emissions (P); (2) ozone oxidized emissions (PO); (3) oxidized emissions + secondary organic aerosol (SOA) (POS); (4) neutralized oxidized emissions + SOA (PONS); and (5) control scenarios: oxidized emissions + SOA in the absence of primary particles (OS), oxidized emissions alone (O), and SOA alone (S). Immediately after 6 hours of exposure, CL in the lung and heart was measured. Tissues were also assayed for thiobarbituric acid reactive substances (TBARS). Exposure to P or PO aerosols led to no changes compared to filtered air in lung or heart CL at any individual plant or when all data were combined. POS caused significant increases in lung CL and TBARS at only one plant, and not in combined data from all plants; PONS resulted in increased lung CL only when data from all plants were combined. Heart CL was also significantly increased with exposure to POS only when data from all plants were combined. PONS increased heart CL significantly in one plant with TBARS accumulation, but not in combined data. Exposure to O, OS, and S had no CL effects. Univariate analyses of individual measured components of the exposure atmospheres did not identify any component associated with increased CL. These data suggest that coal-fired power plant emissions combined with other atmospheric constituents produce limited pulmonary and cardiac oxidative stress.     

Bone metabolism of male rats chronically exposed to cadmium

Recently, based on a female rat model of human exposure, we have reported that low-level chronic exposure to cadmium (Cd) has an injurious effect on the skeleton. The purpose of the current study was to investigate whether the exposure may also affect bone metabolism in a male rat model and to estimate the gender-related differences in the bone effect of Cd. Young male Wistar rats received drinking water containing 0, 1, 5, or 50 mg Cd/l for 12 months. The bone effect of Cd was evaluated using bone densitometry and biochemical markers of bone turnover. Renal handling of calcium (Ca) and phosphate, and serum concentrations of vitamin D metabolites, calcitonin, and parathormone were estimated as well. At treatment with 1 mg Cd/l, corresponding to the low environmental exposure in non-Cd-polluted areas, the bone mineral content (BMC) and density (BMD) at the femur and lumbar spine (L1-L5) and the total skeleton BMD did not differ compared to control. However, from the 6th month of the exposure, the Z score BMD indicated osteopenia in some animals and after 12 months the bone resorption very clearly tended to an increase. The rats' exposure corresponding to human moderate (5 mg Cd/l) and especially relatively high (50 mg Cd/l) exposure dose- and duration-dependently disturbed the processes of bone turnover and bone mass accumulation leading to formation of less dense than normal bone tissue. The effects were accompanied by changes in the serum concentration of calciotropic hormones and disorders in Ca and phosphate metabolism. It can be concluded that low environmental exposure to Cd may be only a subtle risk factor for skeletal demineralization in men. The results together with our previous findings based on an analogous model using female rats give clear evidence that males are less vulnerable to the bone effects of Cd compared to females.     

Changes of serum sex hormone levels and MT mRNA expression in rats orally exposed to cadmium

It has been demonstrated that cadmium (Cd) is carcinogenic to rodent prostate. However, the mechanism of its toxicity is far from fully understood. In the present study, the effects of oral Cd exposure (0, 50, 100, 200 ppm in drinking water) on serum sex hormone levels, the expression of MT-I and MT-II mRNA, and the zinc content of rat prostate were assessed. With Cd administration, serum testosterone (T) levels significantly increased in all Cd groups after 3 months and in the 200 ppm Cd group after 6 months. A significant depression in the serum luteinizing hormone (LH) level was seen in the Cd group (200 ppm) after 6 months. It was noted that Cd administration resulted in a significant down-regulation in the expression of MT-I and MT-II mRNA in the rat ventral prostate. However, no Cd-induced changes in the mRNA expression of Metallothioneins (MTs) were detected in the dorsolateral prostate. After Cd administration, the content of Cd in both the ventral and dorsolateral lobes of the prostate significantly increased with increasing dose and duration of Cd administration. In contrast, the Zn content decreased with Cd administration in both the ventral and dorsolateral lobes of the rat prostate. Taken together, these results suggest that oral Cd exposure may disrupt endocrine homeostasis, changing the distribution of Zn and the mRNA expression of MTs in rat prostate, and that such Cd-induced changes may contribute to the susceptibility of prostate to the carcinogenicity of this heavy metal.     

Ligustrazine modulates renal cysteine biosynthesis in rats exposed to cadmium

The objective of this study was to determine the effect of ligustrazine (TMP) on cadmium (Cd)-induced nephrotoxicity and its relevant mechanism. TMP (50 mg/kg) was injected intraperitoneally (i.p.) into rats 1 h prior to CdCl₂ exposure (at a Cd dose of 0.6 mg/kg). TMP reversed Cd-induced nephrotoxicity, evidenced by the relatively normal architecture of the renal cortex. Additionally, TMP alleviated renal oxidative stress of rats that were exposed to Cd, evidenced by the decreased levels of malondialdehyde (MDA), 4-hydroxynonenal (4-HNE), elevated levels of glutathione (GSH) and GSH/GSSG (glutathione disulfide) ratios. Furthermore, TMP also raised the decreased levels of S-adenosylmethionine (SAM) and cystathionine involved in cysteine biosynthesis in rats exposed to Cd. Further analysis revealed that TMP treatment upregulated expression of several proteins involved in cysteine biosynthesis including methionine adenosyltransferases (MATs) and cystathionine-beta-synthase (CBS). Taken together, these results suggest that TMP remodeled metabolomics of cysteine biosynthesis in rat kidneys and attenuated Cd-induced nephrotoxicity.     

The effect of ozone on the expression of metallothionein in tissues of rats chronically exposed to cadmium

Our aims were to evaluate the expression of metallothionein (MT) in an experimental rat model which experienced chronic exposure to cadmium (Cd) and to measure its expression after ozone therapy (OT) or oxygen (Ox) in the same model, as compared to the control group, which was exposed to neither cadmium nor ozone.Forty male Wistar rats were divided into 5 groups: control, Cd, Cd and Ox, Cd and Oz, and Oz. During our research, Cd concentration (ASA) and MT concentration (ELISA) were determined in supernatants of the kidneys, liver and pancreas. SDS-PAGE analyses and immunohistochemical localization were used to evaluate the level of MT expression in the tissue. In rats intoxicated with Cd, the highest concentration of both Cd and MT was observed in the kidneys and liver, with a significantly lower concentration measured in the pancreas. Ozone therapy reduces the accumulation of cadmium in the liver and kidneys, resulting in a reduced expression of metallothionein in those tissues.     

Disorders in bone metabolism of female rats chronically exposed to cadmium

The effect of cadmium (Cd) on bone metabolism during skeletal development and maturity was investigated on a rat model of human exposure. Young female Wistar rats were exposed to 1, 5, or 50 mg Cd/l in drinking water for 3, 6, 9, and 12 months. Total bone mineral density (T-BMD), bone mineral content (BMC), density (BMD), and bone area at the femur and lumbar spine (L1-L5) were measured densitometrically. Alkaline phosphatase (ALP) and osteocalcin (OC) as bone formation markers, and carboxy-terminal cross-linking telopeptides of type I collagen (CTX) in bone (trabecular and cortical) or serum as bone resorption markers were measured. Renal calcium (Ca) handling and Cd body burden were evaluated as well. At the stage of intensive skeletal development (the first 6 months of the experiment), at all exposure levels, Cd inhibited the processes of bone formation and as a result disturbed the accumulation of bone mass leading to osteopenia (- 1 > Z score/T score BMD > -2.5) and at 5 and 50 mg Cd/l even to more advanced disorders in the BMD. Continuation of the exposure up to skeletal maturity led to high bone turnover with increased resorption enhancing the prevalence of osteopenia or the BMD values having the Z score/T score < -2.5. The results allow for the conclusion that chronic, even low-level exposure to Cd disturbs bone metabolism during skeletal development and maturity by affecting bone turnover most probably through a direct influence on bone formation and resorption, and indirectly via disorders in Ca metabolism. Our findings confirm the hypothesis that environmental exposure to Cd may be a risk factor for low BMD.     

Potentiation of diabetic glomerulopathy in uninephrectomized rats subchronically exposed to cadmium

The renal effects of diabetes mellitus and cadmium (Cd), separately or in combination, were investigated in unilaterally nephrectomized female Sprague-Dawley rats. Diabetes was induced by injection of streptozotocin and Cd was administered in drinking water at a concentration of 100 p.p.m. for 2.5 months. Cd did not affect the reduction in glomerular filtration rate or the rise in beta 2-microglobulinuria caused by diabetes. By contrast, the effect of diabetes on the urinary excretion of albumin, transferrin or IgG was greatly enhanced by concomitant exposure to Cd. This interaction occurred at Cd levels in the renal cortex which are very similar to those found in the general population of industrialized countries. These observations, in agreement with the results of a recent epidemiological study, suggest that Cd polluting the environment might potentiate the development of diabetic nephropathy.     

Extracellular-superoxide dismutase expression in COS7 cells exposed to cadmium chloride

Cadmium (Cd), an industrial and environmental pollutant, preferentially accumulates in the kidney, a major target for Cd-related toxicity. It has been reported that Cd exposure produces reactive oxygen species (ROS) and induces cytotoxicity. Extracellular-superoxide dismutase (EC-SOD) is an antioxidant enzyme that protects the cells from damaging effects of ROS; however, the effect of Cd on the expression of EC-SOD in COS7 cells remains unclear. In this study, exposure to cadmium chloride (CdCl?) enhanced intracellular ROS generation and induced COS7 cell death. Moreover, exposure to Cd decreased the expression of EC-SOD at mRNA and protein levels, but not of other SOD isozymes, copper-and zinc-containing SOD and manganese-containing SOD. The reduction of EC-SOD and cell viability was partially attenuated by pretreatment with an antioxidant, N-acetylcysteine. Further, we determined the involvement of p38-mitogen-activated protein kinase (p38-MAPK) in the reduction of EC-SOD. From these observations, p38-MAPK signaling cascades activated by ROS play a pivotal role in the reduction of EC-SOD, and it is concluded that the reduction of EC-SOD leads to a decrease in the resistance to oxidative stress of Cd-exposed COS7 cells.     

Significance of increase in urinary metallothionein of rats repeatedly exposed to cadmium

Cadmium chloride was injected subcutaneously (s.c.) into female Wistar rats at a dose of 1 mg Cd/kg body weight, 5 times a week up to 10 weeks. At specified intervals, 24-h urine was collected and the excreted amounts of metallothionein (MT), cadmium, copper, zinc and several indicators of renal damage were determined. Concentrations of cadmium and MT in the livers and kidneys of rats were also determined. Both cadmium and MT in the livers and kidneys were increased upon cadmium exposure. The urinary MT excretion started to increase within a week after the start of exposure. This increased excretion preceded those of enzymes and total protein as well as histopathological abnormalities in the proximal tubular cells. After the occurrence of tubular damage that disturbs reabsorption of MT, MT in urine was drastically increased. These results indicate that urinary MT levels may be an indicator not only of cadmium exposure but also of tubular damage.     

Social memory deficits in adult male rats exposed to cadmium in infancy

In two experiments infant rats were injected subcutaneously with 0, 1, or 2 mg Cd/kg on Day 5 or 6 after birth. In adulthood (150 days of age) subjects in both experiments who received the 2 mg/kg dose failed to learn the identity of a strange rat in a social recognition test. Cadmium-treated rats investigated familiar and strange rats equally, whereas control subjects investigated familiar rats much less than unfamiliar individuals. Results with rats in the 1 mg/kg group were less consistent; in Experiment 1 they failed to learn the identity of a stranger, in Experiment 2 they behaved like controls. The level of investigation of a strange rat did not differ among the experimental groups, indicating cadmium did not cause a performance deficit. The 2 mg/kg dose of cadmium had no effect on body weight in Experiment 1 and a small (6.98%), but significant, depressant effect on body weight in Experiment 2. Cadmium exposure in infancy appears to affect social memory processes long after the treatment period.     

Enhanced analgesic response to morphine in adult rats exposed to morphine prenatally

Morphine exposure during development has been shown to produce fetal tolerance to morphine as measured by spontaneous activity only if a particular injection schedule is used. The present study was undertaken to compare the morphine-induced analgesic response in adult offspring of rats which had been injected during the last half of gestation on schedules known to produce fetal tolerance (5 mg/kg morphine at 6 hour intervals) versus a schedule known not to produce fetal tolerance (10 mg/kg morphine at 12 hour intervals). At 30 days postnatally the offspring of animals injected on these 2 schedules show no changes in their responsiveness to the analgetic effect of morphine as determined in the hot-plate test. The present study shows that adult offspring of mothers injected with 20 mg/kg/day of morphine in four divided doses on days 12-20 of gestation have an enhanced analgetic response to morphine in the tail-flick test. In contrast, offspring of mothers injected during the same period of gestation with 20 mg/kg/day of morphine in two divided doses respond to the analgetic effect of morphine in the same manner as the offspring of saline-treated mothers. These results show that the schedule for prenatal morphine administration can play a role in the behavioral effects of morphine in adulthood.     

Comparative analysis of pulmonary irritation by measurements of Penh and protein in bronchoalveolar lavage fluid in brown Norway rats and Wistar rats exposed to irritant aerosols

The object of this study was to compare in two strain of rats (Brown Norway and Wistar) that were acutely exposed (1 x 6 hrs) to aerosols of polymeric methylenediphenyl-4,4'-diisocyanate (MDI), the relative sensitivity of the functional endpoint "enhanced pause (Penh)" with endpoints in bronchoalveolar lavage fluid (BALF) indicative of lower respiratory tract irritation. This approach included an analysis of both the concentration-response and the time-course of effects. Penh was measured repeatedly prior to (baseline) and during a stepped methacholine (MCh) bronchoprovocation challenge in barometric plethysmographic chambers on postexposure days 1, 3, and 7. Results show that total protein in BALF was a sensitive endpoint to probe early effects caused by exposure to irritant polyisocyanate aerosol. Baseline Penh increased in a concentration-dependent manner and paralleled closely the magnitude of increased BALF-protein on postexposure day 1. The time-course of changes in Penh complemented those of BALF-protein observed in previous studies. The incremental increase of Penh during a stepped MCh-challenge was attenuated with increasing baseline Penh values. In summary, these data show that the exposure to irritant concentrations of this aerosolized polyisocyanate caused a transient perturbation of the blood/air-barrier, which can suitably be probed by measurements of protein in BALF. Although Penh appears to parallel these changes, it displays substantially greater variability. The increase in Penh is apparently more related to changes in breathing patterns either caused by stimulation of receptors or changes in the mechanical properties of lung parenchyma. In regard to Penh and BALF-protein, both strains of rats were equally susceptible, although some endpoints displayed a higher variability in Brown Norway rats when compared to Wistar rats. This supports the conclusion that Wistar rats afford a higher diagnostic resolution than BN rats.     

Sensorimotor developmental delays and lower anxiety in rats prenatally exposed to cadmium

Pregnant rats were treated with 0.3 or 0.6 mg cadmium (CdCl2) kg(-1) injected subcutaneously on a daily basis from gestational day 7 to day 15 (organogenesis period). One control group was not injected and the other received saline. Offspring were tested for ontogeny of sensorimotor development and at 45 or 90 days of age for anxiety behavior. The study of sensorimotor development showed that gestational exposure to 0.6 mg Cd kg(-1) produced a delay in the development of the righting reflex and of the cliff aversion in the pups. No differences were observed in the development of the negative geotaxis, nor in the ages of eye and ear opening. Anxiety studies using an elevated plus maze showed a lower anxiety in all the offspring prenatally exposed to 0.6 mg Cd kg(-1) as these rats spent more time and entered the open arms more times compared with those of the other groups. The results demonstrate that exposure to low levels of Cd during organogenesis may modify some central nervous system functions.     

Enhanced acquisition of cocaine self-administration in rats developmentally exposed to lead.

The rate of acquisition of drug self-administration may serve as a predictor of later drug-taking behavior, possibly influencing the vulnerability to use drugs. The present study examined the effects of perinatal (gestation/lactation) lead exposure on adult rates of acquisition of intravenous cocaine self-administration using an automated procedure that included both Pavlovian and operant components. For Experiment 1, female rats were gavaged daily with 0 or 16 mg lead for 30 days prior to breeding with nonexposed males. Metal administration continued through pregnancy and lactation and was discontinued at weaning (postnatal day (PND) 21). Animals born to control or lead-exposed dams subsequently were tested daily as adults in a preparation where sessions included an initial 3-h autoshaping period followed by a 3-h self-administration period where 0.20 mg/kg cocaine was delivered contingently. During autoshaping, intravenous cocaine infusions were paired with the extension and retraction of a lever, while infusions occurred during self-administration only when a lever press was executed (FR-1). The criterion for acquisition was a 2-day period during which a mean of 50 infusions/session occurred during self-administration. Animals were given 35 days to reach criterion. In Experiment 1, accelerated rates of acquisition of cocaine self-administration were evident for lead-exposed animals relative to controls. Overall, the number of self-administered cocaine infusions per session was significantly higher for lead-exposed rats as compared to control rats. Experiment 2 replicated Experiment 1 except that a higher dose of cocaine (0.80 mg/kg) was employed as the reinforcer, and 30 infusions/session was the set criterion. At the higher cocaine dose (Experiment 2), acquisition rates for control and lead-exposed animals were not markedly different, and significantly different infusion rates were not observed.     

Enhanced pulmonary epithelial replication and axial airway mucosubstance changes in F344 rats exposed short-term to mainstream cigarette smoke

Cigarette smoking is associated with respiratory diseases that may be caused by injury to specific pulmonary cells. The injury may manifest itself as site-specific enhanced cellular replication. In this study, rats were exposed either to mainstream cigarette smoke (CS; 250 mg total particulate matter/m(3)) or to filtered air (FA) for 6 h/day, 5 days/week, for 2 weeks. In one group, cells in S-phase were labeled over 7 days by bromodeoxyuridine (BrdU) released from implanted osmotic pumps (pump labeled), while another group received BrdU by injection 2 h prior to necropsy (pulse labeled). Morphometry showed that the type II epithelial BrdU labeling index (LI) was significantly elevated in the CS-exposed animals of both labeling groups. The axial airway and terminal bronchiolar LIs were enhanced by CS only in the pump-labeled group. In a third group (pulse labeled), 2 weeks of recovery following exposure to CS allowed a normalization in the type II LI. In the pump-labeled rats, the CS-induced elevation of the type II LI was greater than the LI elevation in conducting airways, suggesting that the parenchyma may have been injured more than the conducting airways. The terminal bronchiolar LI in the pump-labeled group, regardless of exposure, was significantly greater than the axial airway LI. Pump labeling, in contrast to pulse labeling, could therefore discern differences among replication rates of conducting airway epithelium in different regions of the lung. Mucosubstance (MS) within the axial airway epithelium was quantified by morphometry. The CS exposure did not increase the total number of MS-containing cells or the total number of axial airway epithelial cells, but there was a phenotype change in the MS cells. Neutral MS cells (periodic acid-Schiff-positive) were significantly decreased, while acid MS cells (alcian blue-positive) were slightly increased by CS exposure. Either cell replication and differentiation or differentiation alone may have changed the phenotype in the MS cell population.