Multiple antibodies targeting tumor-specific mutations redirect immune cells to inhibit tumor growth and increase survival in experimental animal models.

BACKGROUND: T cell therapy for cancer involves genetic introduction of a target-binding feature into autologous T cells, ex vivo expansion and single large bolus administration back to the patient. These reprogrammed T cells can be highly effective in killing cells, but tumor heterogeneity results in regrowth of cells that do not sufficiently express the single antigen being targeted. We describe a cell-based therapy that simultaneously targets multiple tumor-specific antigens. METHODS: High-affinity polyclonal rabbit antibodies were generated against nine different surface-related tumor-specific mutations on B16F10 cells. Unsorted splenic effector cells from syngeneic mice were incubated with a cocktail of the nine anti-B16F10 antibodies. These 'armed' effector cells were used to treat mice previously inoculated with B16F10 melanoma cells. RESULTS: The cocktail of nine antibodies resulted in dense homogeneous binding to histological sections of B16F10 cells. Five treatments with the armed effector cells and PD1 inhibition inhibited tumor growth and improved survival. Shortening the interval of the five treatments from every three days to every day increased survival. Arming effector cells with the four antibodies showing best binding to B16F10 cells even further increased survival. CONCLUSIONS: This study demonstrates that ex vivo arming a mixed population of immune effector cells with antibodies targeting multiple tumor-specific mutated proteins in conjunction with PD1 inhibition delayed tumor growth and prolonged survival in mice inoculated with an aggressive melanoma. A remarkably low total antibody dose of less than 5 µg was sufficient to accomplish tumor inhibition. Scaling up to clinical level may be feasible.

Combination treatment with Grb7 peptide and Doxorubicin or Trastuzumab (Herceptin) results in cooperative cell growth inhibition in breast cancer cells.

Grb7 has potential importance in the progression of cancer. We have previously identified a novel peptide that binds to the SH2 domain of Grb7 and inhibits its association with several different receptor tyrosine kinases. We have synthesised the Grb7 peptide, G7-18NATE, with two different cell penetrating peptides, Penetratin and Tat. In this study, we have shown that both Penetratin- and Tat-conjugated G7-18NATE peptides are able to inhibit the proliferation of SK-BR-3, ZR-75-30, MDA-MB-361 and MDA-MB-231 breast cancer cells. There was no significant effects on breast cancer MCF-7cells, non-malignant MCF 10A or 3T3 cells. In addition, there was no significant inhibition of proliferation by Penetratin or Tat alone or by their conjugates with arbitrary peptide sequence in any of the cell lines tested. We determined the EC50 of G7-18NATE-P peptide for SK-BR-3 cell proliferation to be 7.663 x 10(-6) M. Co-treatment of G7-18NATE-P peptide plus Doxorubicin in SK-BR-3 breast cancer cells resulted in an additional inhibition of proliferation, resulting in 56 and 84% decreases in the Doxorubicin EC50 value in the presence of 5 x 10(-6) and 1.0 x 10(-5) M G7-18NATE-P peptide, respectively. Importantly, the co-treatment with Doxorubicin and the delivery peptide did not change the Doxorubicin EC50. Since Grb7 associates with ErbB2, we assessed whether the peptide inhibitor would have a combined effect with a molecule that targets ErbB2, Herceptin. Co-treatment of Herceptin plus 1.0 x 10(-5) M G7-18NATE-P peptide in SK-BR-3 cells resulted in a 46% decrease in the Herceptin EC50 value and no decrease following the co-treatment with Herceptin and penetratin alone. This Grb7 peptide has potential to be developed as a therapeutic agent alone, in combination with traditional chemotherapy, or in combination with other targeting molecules.

Cadmium-induced elevations in the gene expression of the regulatory subunit of gamma-glutamylcysteine synthetase in rat lung and alveolar epithelial cells.

The controlled step in de novo glutathione (GSH) synthesis is catalyzed by gamma-glutamylcysteine synthetase (gamma-GCS), a dimeric enzyme consisting of a heavy catalytic subunit (gamma-GCS-HS) and a light regulatory subunit (gamma-GCS-LS). We have previously reported that exposure to cadmium (Cd) induces pulmonary gamma-GCS-HS mRNA and protein, and that these alterations are accompanied by increases in GSH synthesis and its steady-state level. The current study was designed to test the hypothesis that Cd exposure also up-regulates the expression of the regulatory gamma-GCS subunit. By using northern blotting, we have demonstrated that a single Cd aerosol exposure of adult male Lewis rats results in time- and dose-dependent increases in pulmonary levels of gamma-GCS-LS mRNA. Transcripts of gamma-GCS-LS in rat lung are maximally elevated (8-fold) 2 h following Cd inhalation exposure and remain significantly higher than air controls at 24 h. This response is highly correlated with Cd dose, ranging from 0.9 to 5 mg Cd per m(3), and with lung Cd burden. We also observed Cd-induced up-regulation of gamma-GCS-LS mRNA expression in alveolar epithelial cells exposed to Cd in vitro, either acutely or after repeated passaging in Cd-containing medium. The magnitude of the gamma-GCS regulatory subunit induction observed in Cd-treated cells was approximately five times greater than the induction of the catalytic subunit. These modifications in the expression of gamma-GCS subunits may offer protection from Cd toxicity.

Suppressed oxidant-induced apoptosis in cadmium adapted alveolar epithelial cells and its potential involvement in cadmium carcinogenesis.

Apoptosis involves a series of genetically programmed events associated with endonucleolytic cleavage of DNA. This process is triggered by a variety of agents, including oxidants such as hydrogen peroxide (H(2)O(2)) and it plays a key role in eliminating pre-neoplastic cells from the lung. Failure to do so could favor tumor promotion. The current study demonstrated that alveolar epithelial cells, adapted to cadmium (CdCl(2)) by repeated in vitro exposure, exhibit lower levels of H(2)O(2)-induced apoptosis than similarly challenged non-adapted cells. An immunologic assay, measuring cytoplasmic histone-associated DNA fragments, indicated maximal apoptosis 24 h after exposure to 400 microM H(2)O(2). Non-adapted cells showed a 13-fold increase in oxidant-induced apoptosis while Cd-adapted cells had only a 4-fold elevation. A terminal deoxyribonucleotidyl transferase mediated dUTP nick end labeling (TUNEL) method was used to assess the percentage of cells with DNA breaks consistent with apoptosis. Cd-adapted and non-adapted cells that were not exposed to H(2)O(2) did not differ in TUNEL positivity. However, after H(2)O(2) treatment, the percentage of TUNEL positive cells was 4-fold higher in non-adapted cultures than in adapted ones. Suppression of oxidant-induced apoptosis is due, in part, to up-regulation in the gene expression of several resistance factors including metallothioneins (MT-1 and MT-2), glutathione S-transferases (GST-alpha and GST-pi), and gamma-glutamylcysteine synthetase catalytic subunit (gamma-GCS). These steady-state mRNA changes, determined by Northern blotting, were accompanied by increased levels of MT and gamma-GCS protein, GST activity, and glutathione (GSH). Suppressed oxidant-induced apoptosis, resulting at least in part from these response modifications, could leave pre-neoplastic or neoplastic cells alive, favor clonal expansion, and ultimately lead to cancer development.

Cerebral antioxidant status and free radical generation following glutathione depletion and subsequent recovery.

This study was aimed to evaluate the oxidative damage, production of reactive oxygen species and the status of antioxidative defenses following cerebral GSH depletion induced by two classical depletors, diethylmaleate (DEM, 3 mmol/kg, i.p.) and phorone (PHO, 4 mmol/kg, i.p.). The treatment decreased (40-43%) brain glutathione levels at 2 h, followed by a partial recovery at 24 h. Cerebral glutathione depletion by these agents increased the levels of superoxide anion and hydroxyl radical at both the time intervals; however, hydrogen peroxide was high at 24 h only. It also produced a dramatic increase in the protein carbonyls at 2 h but not at 24h, without any significant effect on lipid peroxidation and conjugated diene levels. These rats showed a significantly lowered superoxide dismutase activity both at 2 h and 24 h of exposure, as compared to controls. Glutathione depletion enhanced catalase activity markedly at 2 h, followed by some recovery at 24 h. While Se-independent glutathione peroxidase (GPx) and glutathione S-transferase activities were increased at both 2 and 24 h time intervals, Se-dependent GPx and glucose-6-phosphate dehydrogenase were induced at 2 h only. Glutathione depletion decreased ceruloplasmin and vitamin E levels significantly at 2 h. However, ascorbic acid remained unaffected. It may be concluded that an acute cerebral glutathione depletion generates higher levels of reactive oxygen species, which may be responsible for oxidative modification of proteins. Some of these changes appear to recover soon after an activation of a variety of cellular antioxidant defense mechanisms and glutathione restoration. It appears that central nervous system is highly vulnerable to oxidative damage following a moderate glutathione depletion that may result from certain diseases or xenobiotic exposures.

Enhanced expression of pulmonary gamma-glutamylcysteine synthetase heavy subunit in rats exposed to cadmium aerosols.

This investigation sought to determine the effect of cadmium (Cd) aerosol exposure on the pulmonary expression of the heavy subunit (HS) of gamma-glutamylcysteine synthetase (gamma-GCS), the rate-limiting enzyme in de novo synthesis of glutathione (GSH). Using Northern hybridization analysis, we demonstrated that CdO inhalation caused time- and dose-dependent increases in the steady-state levels of gamma-GCS-HS mRNA that were highly correlated with lung Cd burden. Observed increases in gamma-GCS-HS gene expression were maximal 2 h following a single aerosol exposure to Cd and appeared to be triggered by an oxidant stress, characterized by a decline in the reduced to oxidized glutathione ratio. Immunoblotting of proteins in lung extracts from treated and untreated animals produced a single protein band corresponding to a molecular weight of 73 kDa. Elevated levels of gamma-GCS-HS mRNA and gamma-GCS-HS protein in lungs of Cd-exposed animals were also accompanied by higher gamma-GCS enzymatic activity and elevations in glutathione (GSH). Immunohistochemical and in situ hybridization studies were used to identify compartments in the lung where Cd-induced expression of gamma-GCS-HS was localized. The most prominent staining for gamma-GCS-HS protein and gamma-GCS-HS mRNA was observed in the alveolar epithelium of Cd-exposed animals. Quantitative image analysis confirmed a good agreement between relative levels of protein and mRNA transcripts for gamma-GCS-HS. These observations suggest that resistance to Cd toxicity in the lung may reflect the ability of specific lung cells to upregulate gamma-GCS expression and increase de novo GSH synthesis as an adaptive response.

Cadmium-mediated oxidative stress in alveolar epithelial cells induces the expression of gamma-glutamylcysteine synthetase catalytic subunit and glutathione S-transferase alpha and pi isoforms: potential role of activator protein-1.

Exposure of rat alveolar epithelial cells to 10 micromol/L CdCl2 causes time-dependent increases in steady-state mRNA levels of the gamma-glutamylcysteine synthetase catalytic (heavy) subunit (gamma-GCS) and of glutathione S-transferase isoforms (GST-alpha and GST-pi). The expression of gamma-GCS was significantly increased as early as 2 h after addition of cadmium. Maximal induction of gamma-GCS mRNA (approximately 4-fold), at 8 h, was subsequently followed by increases in gamma-GCS activity/protein and glutathione (GSH) levels. Maximal elevations in GST-pi (approximately 2-fold) and GST-alpha (approximately 10-fold) transcripts, at 8 and 24 h, respectively, were also accompanied by enhanced GST activity. Cadmium-induced oxidative stress, assessed by alterations in GSH homeostasis and an accelerated rate of intracellular oxidant production, could constitute early events in the signal transduction pathway mediating these responses. The dimeric transcription factor, activator protein-1 (AP-1), may also play a regulatory role in this process. This association is suggested by transcriptional activation of the immediate-early response genes, c-fos and c-jun, within 15 min after exposure to cadmium and by the enhancement of AP-1 DNA binding activity, involving a c-Jun protein complex, which is maximally induced (approximately 4-fold) by 2 h. These molecular changes likely function together to protect alveolar epithelial cells against cadmium toxicity.

Potential role of cerebral glutathione in the maintenance of blood-brain barrier integrity in rat.

Using the model of glutathione (GSH) depletion, possible role of GSH in the maintenance of blood-brain barrier (BBB) integrity was evaluated in rats. Administration (i.p.) of GSH depletors, diethyl maleate (DEM, 1-4 mmol/kg), phorone (2-3 mmol/kg) and 2-cyclohexene-1-one (CHX, 1 mmol/kg), to male adults was found to deplete brain and liver GSH and increase the BBB permeability to micromolecular tracers (sodium fluorescein and [14C]sucrose) in a dose-dependent manner at 2h. However, BBB permeability to macromolecular tracers such as horseradish peroxidase and Evan's blue remained unaltered. It was also shown that observed BBB permeability dysfunction was associated with brain GSH depletion. A lower magnitude of BBB increase in rat neonates, as compared to adults, indicated a possible bigger role of GSH in the BBB function of mature brain. The treatment with N-acetylcysteine, methionine and GSH provided a partial to full protection against DEM-induced brain (microvessel) GSH depletion and BBB dysfunction; however, the treatment with alpha-tocopherol, ascorbic acid and turmeric were not effective. Our studies showed that cerebral GSH plays an important role in maintaining the functional BBB integrity.

Developmental pattern of reactive oxygen species generation and antioxidative defense machinery in rat cerebral microvessels.

The developmental profile of certain enzymatic antioxidants as well as the generation of reactive oxygen species was studied in the rat cerebral microvessels during first three weeks of life and the levels were compared to those present in adults. The data showed a higher generation of superoxide anion (+67%) and H2O2 (+200%) at postnatal day (PND) 21. Superoxide anion production was significantly lower (-24%) at PND 14 and almost comparable to adult values at PND 7. The activity of superoxide dismutase increased with development and attained an adult level at PND 21. Catalase was higher in neonates with a maximum activity at PND 7 and 14 (+68, 69%). The measurement of microvessel glutathione and glutathione-related antioxidant enzymes showed that glutathione level was higher at PND 7, which declined to an adult level at PND 14. Se-dependent GPx showed a marked increase between PND 14 and 21, however, it declined in adults. The activity of Se-independent glutathione peroxidase was very low in cerebral microvessels. Glutathione reductase activity in 7-day-old, that was comparable to adult level, declined at PND 14 and 21. The level of glutathione S-transferase was higher (+43%) at PND 21. The activity of microvessel marker enzyme gamma-glutatmyl transpeptidase increased with age, whereas, alkaline phosphatase showed a slight increase up to PND 14 and thereafter it declined. Lipid peroxidation was found to be significantly lower (-18%) at PND 21 as compared to adults. It may be concluded that developing cerebral microvessels contain high levels of several antioxidant enzymes that are more or equal to those present in adult brain microvessels.

Characterization of cadmium-induced apoptosis in rat lung epithelial cells: evidence for the participation of oxidant stress.

The mode of cadmium-induced cell death was investigated in a rat lung epithelial cell line. Cells, grown to near confluence, were exposed to 0-30 microM CdCl2 for 0-72 h. Phase contrast microscopy and fluorescent nuclear staining showed that Cd caused morphological alterations in lung epithelial cells that are characteristic of apoptosis. These changes included cell shrinkage, detachment of the cell from its neighbors, cytoplasmic and chromatin condensation, and fragmentation of the nucleus into multiple chromatin bodies surrounded by remnants of the nuclear envelope. Apoptotic DNA degradation was validated and quantitated using a sensitive enzyme-linked immunosorbent assay (ELISA) which measures the amount of histone-bound DNA fragments in the cytosol. Using this technique, a maximum level of apoptosis (5-fold higher than control) was observed in cultures exposed for 48 h to 20 microM CdCl2. The terminal deoxyribonucleotidyl transferase mediated dUTP nick end labeling method (TUNEL) was subsequently used to determine the percentage of cells that contained Cd-induced DNA strand breaks. After 48 h, approximately 54% of the cells exposed to 20 microM Cd were TUNEL positive compared to less than 2% for control cells. Although the mechanisms by which Cd initiates apoptosis in these cells are presently not known, reactive oxygen species are likely to play a role. This possibility is supported by the finding that the first morphological features indicative of apoptosis were preceded by the up-regulation of oxidant stress genes (glutathione S-transferase-alpha, gamma-glutamylcysteine synthetase, and metallothionein-1), activation of redox sensitive transcription factors (AP-1 and NF-kappaB), and changes in various forms of glutathione (reduced, oxidized, and protein-bound).

Glutathione depletion and oxidative damage in mitochondria following exposure to cadmium in rat liver and kidney.

The dose-dependent effects of cadmium (Cd) on mitochondria and post-mitochondrial supernatant (PMS) of liver and kidney were investigated in adult male albino rats. Two groups of rats were injected intraperitoneally with 0.1 mg Cd/kg body weight and 1 mg/kg body weight, respectively, for a period of 3 months (5 days/week). This resulted in a significant decrease in total glutathione (GSH) levels, irrespective of the doses, in mitochondrial as well as in PMS fractions of liver and kidney. In contrast, end products of lipid and protein were significantly increased in a dose-dependent manner in subcellular fractions of liver and kidney. These results suggest that the depletion of tissue glutathione levels is not a primary reason of the observed oxidative damage in liver and kidney caused by Cd.

Functional impairment of blood-brain barrier following pesticide exposure during early development in rats.

1. The effect of certain pesticides on the functional integrity of the developing blood-brain barrier (BBB) was studied following single and repeated exposure, and after subsequent withdrawal in rats. 2. Ten-day-old rat pups exposed orally to quinalphos (QP, organophosphate), cypermethrin (CM, pyrethroid) and lindane (LD, organochlorine) at a dose of 1/50th of LD50, showed a significant increase in the brain uptake index (BUI) for a micromolecular tracer, sodium fluorescein (SF), by 97, 37 and 72%, respectively, after 2 h. Residual increases in the BUI were found even after 3 days of the single treatment of QP (28%) and LD (23%). 3. Repeated exposure for 8 days (postnatal days (PND) 10-17) with QP, CM and LD increased the BBB permeability by 130, 80 and 50%, respectively. Recovery from these changes was complete in QP and LD-treated animals after 13 days (PND 18-30) of withdrawal. However, CM showed persistent effects that were normalized only after 43 days (PND 18-60) of withdrawal. 4. A single dose reduced to 1/100th of LD50 also increased BUI in 10-day-old rat pups following QP (20%) and CM (28%) exposure at 2 h. 5. An age-dependent effect of these pesticides was evident from the study showing higher magnitude of BUI changes in 10-day-old rats as compared to that in 15-day-old rats. Furthermore, adult rats did not show any effect on BBB permeability even at a higher dose (1/25th of LD50) of these pesticides given alone or in combination with piperonyl butoxide (600 mg/kg, i.p.) for 3 consecutive days. 6. This study showed that developing BBB is highly vulnerable to single or repeated exposure of certain pesticides. The observed persistent effects during brain development even after withdrawal of the treatment may produce some neurological dysfunction at later life as well.

Profile of reactive oxygen species generation and antioxidative mechanisms in the maturing rat kidney.

The antioxidative potential and reactive oxygen species generation were assessed in rat kidney during early critical periods of development and maturation. Superoxide anion generation was found to be low in kidney during early postnatal days of development, whereas hydrogen peroxide levels remained unaltered during development. The levels of thiobarbituric acid reactive substances and protein carbonyls in developing kidney were higher during early postnatal days, up to 26 days after birth, compared to the adult levels. Kidney sulphydryl contents were significantly less during early periods (9 days postnatally) of development compared to adults but attain adult value by postnatal day 26. The levels of ascorbic acid and ceruloplasmin were also higher in developing kidney than in adults. Among enzymic antioxidants, the levels of glutathione peroxidase (GPx) enzyme in developing kidney were high during the early developmental period of the study as compared to adults; however, superoxide dismutase (SOD), catalase (CAT) and glutathione reductase (GR) were found to be significantly low at early postnatal days up to 16 days of age, which subsequently attained maturational level by the age of 26 days. The levels of antioxidant enzymes and sulphydryl contents in the developing kidney during early periods after birth are low but they increase subsequently with increasing age. Therefore, the present finding suggests that immature kidneys are in a highly dynamic stage of development during the early period and are equipped with antioxidative defence mechanisms that may have a predominant role in protecting against oxidative challenge.

Effect of pyrethroid-based liquid mosquito repellent inhalation on the blood-brain barrier function and oxidative damage in selected organs of developing rats.

Pesticides have been implicated in various neurological disorders in humans and experimental animals. Our earlier studies have demonstrated a high vulnerability of developing blood-brain barrier (BBB) towards very low level exposure of quinalphos, cypermethrin and lindane. Earlier it has been observed that a cypermethrin-induced increase in the BBB permeability of neonatal rats was found to be persistent, requiring a longer period of withdrawal for complete recovery. These observations lead us to investigate the effect of a commonly available liquid mosquito repellent (MR) containing a pyrethroid compound, allethrin (3.6% w/v), on the functional integrity of the developing BBB and on certain parameters of oxidative damage in brain, liver and kidney. Two-day-old rat pups were allowed to inhale the MR (18 h per day) for 8 days (postnatal days (PND) 2-9). Rats exposed to MR were further withdrawn from the exposure for 8 days (PND 10-17) to study whether the changes induced following inhalation are reversible. Results of the study have shown a significant increase in the BBB permeability (45%) of the MR-exposed rat pups to a micromolecular tracer, sodium fluorescein (mol. wt. 376), used for the quantitative assessment of the BBB permeability, suggesting a delayed maturity of the BBB system. Brain glutathione (GSH) levels were also decreased (17%) in the exposed individuals. The oxidatively damaged end-products of lipids, measured as lipid hydroperoxides and conjugated dienes, were found to be increased in brain (42%, 16%), liver (34%, 20%) and kidney (68%, 29%), respectively. The oxidative product of protein, measured as protein carbonyls, was also increased significantly in liver (43%) and kidney (16%) of the MR-exposed rat pups as compared to age-matched controls. The biochemical changes that occurred in the BBB permeability and the oxidatively damaged end-products following MR inhalation in neonatal rats were, however, found to be completely recovered except for an increase in brain GSH (28%) level. The results suggest the possibility of health risk due to exposure to pesticide-based mosquito repellents, especially when exposure takes place in individuals at an early age.

Effects of neonatal quinalphos exposure and subsequent withdrawal on free radical generation and antioxidative defenses in developing rat brain.

Ten-day-old rat pups were given quinalphos (QP, 0.5 mg kg(-1)) orally up to postnatal days (PND) 21 or 45. A group of rats exposed to QP was withdrawn from the treatment at PND 21 and was killed at PND 45 for the withdrawal study. Acetylcholinesterase decreased in the brain and blood after QP exposure but recovered after withdrawal. Superoxide radical generation in brain was increased by 43%, 59% and 39% following exposure up to PND 21 and 45 and in the withdrawal group, respectively. Quinalphos did not alter hydrogen peroxide formation but increased hydroxyl radical (19%) production at PND 45. Quinalphos elevated the activities of superoxide dismutase by 30%, (although the increase from 0.24 to 0.31 was physiologically not significant) and catalase by 50% at PND 21, which up on withdrawal of the exposure at PND 22 recovered partially or completely at PND 45. However, the continuous exposure up to PND 45 decreased superoxide dismutase (63%) and catalase (31%) activities. Selenium-independent glutathione peroxidase (GPx) was increased at PND 21 (31%) and PND 45 (152%) after QP exposure and there was complete recovery after withdrawal. Selenium-dependent GPx, which was elevated slightly at PND 21, was also normalized after withdrawal. Prolonged exposure to QP did not alter the ascorbic acid content and produced a marked decrease in cerruloplasmin (46%) levels. Brain glutathione levels increased marginally in QP-exposed rats and became normal in the withdrawal group. Quinalphos exposure up to PND 45 enhanced brain lipid peroxidation (28%) and decreased vitamin E levels (15%). It appears that neonatal QP exposure produces cerebral oxidative stress, which may result in deleterious effects on central nervous system function immediately and in later life.

Cytochrome P450-dependent oxidation and glutathione conjugation of xenobiotics in alloxan-induced diabetic rat.

The status of cytochrome P450-dependent oxidative biotransformation of aminopyrine and benzo(a)pyrene (Phase I reaction) and glutathione S-transferase (GST) catalyzed conjugation with 1-chloro-2,4-dinitrobenzene (CDNB) (Phase II reaction) was evaluated in diabetic rats sacrificed 3 weeks after alloxan treatment (2 doses of 75 mg/kg at an interval of 48 h, i.p.). Alloxan treatment caused 3-4 fold increase in blood glucose level and 68% rise in glycosylated hemoglobin content. There were significant decreases in the activities of the hepatic aminopyrine N-demethylase and aromatic hydrocarbon hydroxylase (AHH) in diabetic rats as compared with the controls. The activity of GST was also significantly reduced in liver and kidney, whereas remained unchanged in the brain. These results suggest that a prolonged diabetic state depresses the metabolism of xenobiotics and probably of some endogenous compounds as well in liver and kidney.

Ontogenic profile of brain lipids following perinatal exposure to cadmium.

Uniparous female rats were exposed to 10 ppm cadmium (as Cd(CH3COO)2) in drinking water, ad libitum, from day 0 of pregnancy; neonates exposed to Cd gestationally and lactationally were separated from their mothers at 21 days of age and were given 10 ppm Cd in drinking water up to 45 days of age. The sequential analysis of brain lipids during development showed a significant reduction in the total lipid levels at 15, 21, 30 and 45 days of age compared to the respective controls. No change in the triglyceride levels was observed in the Cd-exposed group, except for a slight but statistically significant increase (12%, p < 0.05) at day 15 of postnatal life. Metallic exposure also decreased the cholesterol content of brain at different time intervals in comparison to its levels in age-matched control rats. The analysis of sialic acid content of the total gangliosides revealed elevated levels of this group of glycolipids at 21 (25%), 30 (17%) and 45 (23%) days of age compared to their counterpart controls. However, a remarkable reduction in the concentration of brain galactosylceramide (30-43%) and 3'-sulphogalactosylceramide (24-37%) at 21, 30 and 45 days of age was observed following pre- and postnatal exposure of cadmium in comparison to the respective controls. The ontogenic profile of different brain phospholipids in the Cd-exposed group showed an increase in the levels of phosphatidylethanolamine at 21 (31%), 30 (25%) and 45 (19%) days of age; the phosphatidylcholine contents increased at day 21 (14%), followed by a significant decrease at 30 (14%) and 45 (19%) days compared to age-matched controls. Brain phosphatidylinositol, phosphatidylserine and sphingomyelin did not show any alteration at early periods of exposure but decreased significantly following continued exposure by 34%, 45% and 21%, respectively, at 45 days of age. Metal analysis in the brain showed a reduction in zinc and copper levels at 15 and 21 days of age in Cd-exposed animals; however, iron levels remained comparable with control values throughout the experiment. It may be concluded that an early exposure of Cd may produce alteration in the development of different lipids, which may produce CNS dysfunctions with a possibility of being manifested even in later life.

Cadmium-induced alterations in blood-brain barrier permeability and its possible correlation with decreased microvessel antioxidant potential in rat.

1. Male albino rats of 21 days age were exposed to 10 p.p.m. cadmium (CdCl2 salt) in drinking water, ad libitum, for 90 days. It increased the brain cadmium levels by 76% (P < 0.05) and 165% (P < 0.001) respectively at 30 and 90 days of exposure compared to controls. 2. Cadmium increased blood-brain barrier permeability of fluoroscein dye (24%, P < 0.02) and the levels of brain microvessel malondialdehyde (31%, P < 0.01) at 90 days of exposure. However, these parameters did not alter significantly at 30 days of exposure. 3. Increased activities of microvessel superoxide dismutase (18%, P < 0.02), glutathione peroxidase (20%, P < 0.01) and catalase (28%, P < 0.01) were observed at 30 days of exposure. 4. The continuation of the Cd treatment for 90 days decreased the levels of superoxide dismutase (30%, P < 0.001), glutathione peroxidase (23%, P < 0.005), catalase (25%, P < 0.005), glutathione reductase (18%, P < 0.02), vitamin E (20%, P < 0.01), glutathione (26%, P < 0.01), ascorbic acid (18%, P < 0.05) and ceruloplasmin (13%, P < 0.05) in the microvessal preparation compared to controls. 5. It appears that Cd-induced blood-brain barrier dysfunction may be related to the depletion of microvessel antioxidant substances along with increase in lipid peroxidation at 90 days of exposure.

Maternal cadmium intoxication: effects on fetal brain antioxidant defense parameters.

The antioxidant potential of the brain in developing fetuses was assessed at gestational days (GD) 16, 18 and 20 and postnatal day (PND)1. Higher activities of superoxide dismutase (SOD) and glutathione peroxidase (GPx) were noticed during fetal development which were reduced to about half and one-quarter, respectively, at PND 1. Glutathione reductase (GR) activity remained stationary throughout the experiment and the values were very high compared to those reported for weanling rats. In contrast, catalase (CAT) activity increased with development. Glutathione (GSH) and total sulfhydryls (TSH) were maximum in 16-day fetal brains and declined subsequently. Brain lipid peroxidation (LPO) was found to increase with age. A group of animals was exposed to 20 ppm cadmium (Cd) in drinking water from the day of conception up to PND 1. Cd was found to increase the activities of brain SOD, CAT, and GR significantly at all the time intervals. The metal exposure decreased fetal brain GPx at GD 18 and 20, whereas GPx activity declined precipitously in both groups on PND 1. Cd caused both increments and decrements in the GSH and TSH levels (depending on gestational day) and increased the LPO in brain. It may be concluded that the Cd-intoxicated fetal brain undergoes significant changes in antioxidant defense parameters which, overall, may be sufficient to permit near-normal development and prevent substantial oxidant damage.