The opioid system in the gastrointestinal tract.

Mu-, delta- and kappa-opioid receptors (ORs) mediate the effects of endogenous opioids and opiate drugs. Here we report (1) the distribution of muOR in the guinea-pig and human gastrointestinal tract in relation to endogenous ligands, to functionally distinct structures in the gut and to deltaOR and kappaOR; and (2) the ligand-induced muOR endocytosis in enteric neurones using in vitro and in vivo models. In the guinea pig, muOR immunoreactivity is confined mainly to the myenteric plexus. MuOR myenteric neurones are most numerous in the small intestine, followed by the stomach and the proximal colon. MuOR immunoreactive fibres are dense in the muscle layer and the deep muscular plexus, where they are in close association with interstitial cells of Cajal. This distribution closely matches the pattern of enkephalin. MuOR enteric neurones comprise functionally distinct populations of neurones of the ascending and descending pathways of the peristaltic reflex. In human gut, muOR immunoreactivity is localized to myenteric and submucosal neurones and to immune cells of the lamina propria. DeltaOR immunoreactivity is located in both plexuses where it is predominantly in varicose fibres in the plexuses, muscle and mucosa, whereas kappaOR immunoreactivity appears to be confined to the myenteric plexus and to bundles of fibres in the muscle. MuOR undergoes endocytosis in a concentration-dependent manner, in vitro and in vivo. Pronounced muOR endocytosis is observed in neurones from animals that underwent abdominal surgery that has been shown to induce delay in gastrointestinal transit. We can conclude that all three ORs are localized to the enteric nervous system with differences among species, and that muOR endocytosis can be utilized as a means to visualize enteric neurones activated by opioids and sites of opioid release.

Abdominal surgery induces mu opioid receptor endocytosis in enteric neurons of the guinea-pig ileum.

Immunohistochemistry and confocal microscopy were used to investigate mu opioid receptor (muOR) internalization in enteric neurons of the guinea-pig ileum following abdominal surgery. The following surgical procedures were performed under halothane or isofluorane anesthesia: a) midline abdominal skin incision, b) laparotomy or c) laparotomy with intestinal manipulation. Gastrointestinal transit was evaluated by using a non-absorbable marker and measuring fecal pellet output. In neurons from normal and control (anesthesia alone) animals, muOR was predominantly at the cell surface. muOR endocytosis following skin incision was not significantly different from controls (21.2+/-3.5% vs. 13.7+/-2.1%, mean+/-S.E.M.), whereas it was significantly increased by laparotomy (46.5+/-6.1%; P<0.01 vs. controls) or laparotomy plus intestinal manipulation (40.5+/-6.1%; P<0.01 vs. controls) 30 min following surgery compared with controls. muOR endocytosis remained elevated at 4 h (38.6+/-1.2%; P<0.01 vs. controls), whereas it was similar to controls at 6 and 12 h (17.5+/-5.8% and 11.2+/-3.0%). muOR endocytosis occurred in cholinergic and nitrergic neurons. Gastrointestinal transit was significantly delayed by laparotomy or laparotomy plus intestinal manipulation (12.8+/-1.2 and 13.8+/-0.6 h vs. 7.0+/-0.5 in controls; P<0.01), but was not significantly changed by skin incision (8.2+/-0.6 h). The findings of the present study support the concept that the noxious stimulation caused by abdominal surgery induces release of endogenous opioids thus resulting in muOR endocytosis in neurochemically distinct enteric neurons. muOR internalization can serve as indirect evidence of opioid release and as a means to visualize neuronal pathways activated by opioids.

Ligand-induced mu opioid receptor endocytosis and recycling in enteric neurons.

Immunohistochemistry and confocal microscopy were used to investigate endocytosis and recycling of the native mu opioid receptor (muOR) in enteric neurons. Isolated segments of the guinea-pig ileum were exposed to increasing concentrations of muOR agonists at 4 degrees C to allow ligand binding and warming to 37 degrees C for 0 min (baseline) to 6 h in ligand-free medium to allow receptor internalization and recycling. The endogenous ligand, [Met]enkephalin, and [D-Ala(2),MePhe(4),Gly-ol(5)] enkephalin (DAMGO), an opioid analog, and the alkaloids, etorphine and fentanyl, induced rapid internalization of muOR immunoreactivity in enteric neurons, whereas morphine did not. muOR internalization was prevented by muOR antagonists. Basal levels of muOR immunoreactivity in the cytoplasm were 10.52+/-2.05%. DAMGO (1 nM-100 microM) induced a concentration-dependent increase of muOR immunofluorescence density in the cytoplasm to a maximum of 84.37+/-2.26%. Translocation of muOR immunoreactivity in the cytoplasm was detected at 2 min, reached the maximum at 15-30 min, remained at similar levels for 2 h, began decreasing at 4 h, and was at baseline values at 6 h. A second exposure to DAMGO (100 nM) following recovery of internalized muOR immunoreactivity at the cell surface induced a translocation of muOR immunoreactivity in the cytoplasm comparable to the one observed following the first exposure (46.89+/-3.11% versus 43.31+/-3.80%). muOR internalization was prevented by hyperosmolar sucrose, phenylarsine oxide or potassium depletion, which inhibit clathrin-mediated endocytosis. muOR recycling was prevented by pre-treatment with bafilomycin A1, an acidotropic agent that inhibits endosomal acidification, but not by the protein synthesis inhibitor, cycloheximide. This study shows that native muOR in enteric neurons undergoes ligand-selective endocytosis, which is primarily clathrin-mediated, and recycles following endosomal acidification. Following recycling, muOR is activated and internalized by DAMGO indicating that recycled receptors are functional.

Effects of glutathione on chromium-induced DNA crosslinking and DNA polymerase arrest.

Hexavalent chromium (Cr (VI)) is reduced intracellularly to Cr (V), Cr (IV) and Cr (III) by ascorbate (Asc), cysteine and glutathione (GSH). These metabolites induce a spectrum of genomic DNA damage resulting in the inhibition of DNA replication. Our previous studies have shown that treatment of DNA with Cr (III) or Cr (VI) plus Asc results in the formation of DNA-Cr-DNA crosslinks (Cr-DDC) and guanine-specific arrests of both prokaryotic and mammalian DNA polymerases. GSH not only acts as a reductant of Cr (VI) but also becomes crosslinked to DNA by Cr, thus, the focus of the present study was to examine the role of GSH in Cr-induced DNA damage and polymerase arrests. Co-incubation of Cr (III) with plasmid DNA in the presence of GSH led to the crosslinking of GSH to DNA. GSH co-treatment with Cr (III) also led to a decrease in the degree of Cr-induced DNA interstrand crosslinks relative to Cr (III) alone, without affecting total Cr DNA binding. DNA polymerase arrests were observed following treatment of DNA with Cr (III) alone, but were markedly reduced when GSH was added to the reaction mixture. Pre-formed polymerase-arresting lesions (Cr-DDC) were not removed by subsequent addition of GSH. Treatment of DNA with Cr (VI), in the presence of GSH, resulted in crosslinking of GSH to DNA, but failed to produce detectable DNA interstrand crosslinks or polymerase arrests. The inhibitory effect of GSH on Cr-induced polymerase arrest was further confirmed in human genomic DNA using quantitative PCR (QPCR) analysis. Treatment of genomic DNA with Cr (III) resulted in a marked inhibition of the amplification of a 1.6 kb target fragment of the p53 gene by Taq polymerase. This was almost completely prevented by co-treatment with GSH and Cr (III). These results indicate that Cr-induced DNA interstrand crosslinks, and not DNA-Cr-GSH crosslinks, are the principal lesions responsible for blocking DNA replication. Moreover, the formation of DNA-Cr-GSH crosslinks may actually preclude the formation of the polymerase arresting lesions.

Mechanism of apoptosis and determination of cellular fate in chromium(VI)-exposed populations of telomerase-immortalized human fibroblasts.

The cellular responses to carcinogen exposure influence cellular fate, which in turn modulates the neoplastic response. Certain hexavalent chromium [Cr(VI)] compounds are implicated as occupational respiratory carcinogens at doses that are both genotoxic and cytotoxic. We examined the mechanism of Cr(VI)-induced apoptosis in normal human fibroblasts (BJ) immortalized by human telomerase gene transfection (BJ-hTERT), and we assessed the spectrum of cumulative cellular fates [(a) regaining of replicative potential; (b) terminal growth arrest; or (c) apoptosis] for a narrow range of increasingly genotoxic doses of Cr(VI). Exposure of BJ-hTERT cells to Cr(VI) resulted in a dose-dependent increase in apoptosis that involved mitochondrial disruption as evidenced by mitochondrial membrane depolarization and cytochrome c release. The initial response to Cr(VI) exposure was inhibition of cell cycle progression. At the lowest dose tested (1 microM; 32% clonogenic survival), the cell cycle inhibition led to terminal growth arrest but no apoptosis. The fraction of terminally growth arrested cells increased as the dose was increased to 3 microM but then decreased at 4, 5, and 6 microM as apoptosis became the predominant cell fate. Our results suggest that cell populations exposed to Cr(VI) have a different spectrum of responses, depending on the extent of DNA damage, and that the regaining of replicative potential after relatively higher genotoxic exposures may be attributable to either escape from, or resistance to, terminal growth arrest or apoptosis.

Reversal of physiological stress-induced resistance to topoisomerase II inhibitors using an inducible phosphorylation site-deficient mutant of I kappa B alpha.

Physiological stress conditions associated with the tumor microenvironment play a role in resistance to anticancer therapy. In this study, treatment of EMT6 mouse mammary tumor cells with hypoxia or the chemical stress agents brefeldin A (BFA) or okadaic acid (OA) causes the development of resistance to the topoisomerase II inhibitor etoposide. The mechanism of physiological stress-induced drug resistance may involve the activation of stress-responsive proteins and transcription factors. Our previous work shows that treatment with BFA or OA causes activation of the nuclear transcription factor NF-kappa B. Pretreatment with the proteasome inhibitor carbobenzyoxyl-leucinyl-leucinyl-leucinal inhibits stress-induced NF-kappa B activation and reverses BFA-induced drug resistance. To test whether NF-kappa B specifically mediates stress-induced drug resistance, an inducible phosphorylation site-deficient mutant of I kappa B alpha (I kappa B alpha M, S32/36A) was introduced into EMT6 cells. In this study, we show that I kappa B alpha M expression inhibits stress-induced NF-kappa B activation and prevents BFA-, hypoxia-, and OA-induced resistance to etoposide. These results indicate that NF-kappa B activation mediates both chemical and physiological drug resistance to etoposide. Furthermore, they imply that coadministration of agents that inhibit NF-kappa B may enhance the efficacy of topoisomerase II inhibitors in clinical cancer chemotherapy.

Dynamin and Rab5a-dependent trafficking and signaling of the neurokinin 1 receptor.

Understanding the molecular mechanisms of agonist-induced trafficking of G-protein-coupled receptors is important because of the essential role of trafficking in signal transduction. We examined the role of the GTPases dynamin 1 and Rab5a in substance P (SP)-induced trafficking and signaling of the neurokinin 1 receptor (NK1R), an important mediator of pain, depression, and inflammation, by studying transfected cells and enteric neurons that naturally express the NK1R. In unstimulated cells, the NK1R colocalized with dynamin at the plasma membrane, and Rab5a was detected in endosomes. SP induced translocation of the receptor into endosomes containing Rab5a immediately beneath the plasma membrane and then in a perinuclear location. Expression of the dominant negative mutants dynamin 1 K44E and Rab5aS34N inhibited endocytosis of SP by 45 and 32%, respectively. Dynamin K44E caused membrane retention of the NK1R, whereas Rab5aS34N also impeded the translocation of the receptor from superficially located to perinuclear endosomes. Both dynamin K44E and Rab5aS34N strongly inhibited resensitization of SP-induced Ca(2+) mobilization by 60 and 85%, respectively, but had no effect on NK1R desensitization. Dynamin K44E but not Rab5aS34N markedly reduced SP-induced phosphorylation of extracellular signal regulated kinases 1 and 2. Thus, dynamin mediates the formation of endosomes containing the NK1R, and Rab5a mediates both endosomal formation and their translocation from a superficial to a perinuclear location. Dynamin and Rab5a-dependent trafficking is essential for NK1R resensitization but is not necessary for desensitization of signaling. Dynamin-dependent but not Rab5a-dependent trafficking is required for coupling of the NK1R to the mitogen-activated protein kinase cascade. These processes may regulate the nociceptive, depressive, and proinflammatory effects of SP.

Cyclosporin A inhibits chromium(VI)-induced apoptosis and mitochondrial cytochrome c release and restores clonogenic survival in CHO cells.

A variety of key events in the molecular apoptotic pathway involve the mitochondria. Cyclosporin A (csA) affects the mitochondria by inhibiting the mitochondrial permeability transition (MPT), thereby preventing disruption of the transmembrane potential. The role of the MPT in apoptosis is not fully understood, but inhibition of the MPT may prevent the release of mitochondrial caspase activators, such as cytochrome c (cyt c), into the cytosol. Certain hexavalent chromium [Cr(VI)] compounds are known occupational respiratory tract toxins and carcinogens. We have previously shown that these compounds induce apoptosis as a predominant mode of cell death and that this effect can be observed in cell culture using soluble Cr(VI). We show here that Cr(VI)-induced apoptosis in Chinese hamster ovary (CHO) cells involves disruption of mitochondrial stability. Using a cyt c-specific monoclonal antibody, we observed a dose-dependent release of mitochondrial cyt c in cytosolic extracts of CHO cells exposed to apoptogenic doses of sodium chromate. Co-treatment of these cells with csA inhibited the release of cyt c and abrogated Cr(VI)-induced apoptosis as determined by a reduction in internucleosomal DNA fragmentation. Co-treatment with csA also markedly increased clonogenic survival of Cr(VI)-treated CHO cells. In contrast, the general caspase inhibitor Z-VAD-FMK markedly inhibited most of the morphological and biochemical parameters of apoptosis but did not prevent cyt c release and did not increase clonogenic survival. These results suggest that the MPT plays an important role in the regulation of mitochondrial cyt c release and that this may be a critical point in the apoptotic pathway in which cells are irreversibly committed to death.

Inhibitors of prostaglandin synthesis inhibit human prostate tumor cell invasiveness and reduce the release of matrix metalloproteinases.

Eicosanoids modulate the interaction of tumor cells with various host components in cancer metastasis. Their synthesis involves the release of arachidonic acid (AA) from cellular phospholipids by phospholipase A2 (PLA2), followed by metabolism by cyclooxygenases (COXs) and lipooxygenases (LOXs). This study aimed to identify the pathway(s) of AA metabolism that are required for the invasion of prostate tumor cells. DU-145 and PC-3 human prostate cancer cell lines were used to test the effect of inhibitors of PLA2, COX, or LOX on the invasion of prostate tumor cells through Matrigel in vitro using the Boyden chamber assay and fibroblast-conditioned medium as the chemoattractant. We used nontoxic doses that did not inhibit simple cell motility and did not decrease clonogenic survival. All of the inhibitors caused a significant reduction in AA release from treated cells compared with control cells, which indicated that the treatments were biochemically active. Invasion through Matrigel was inhibited by the PLA2 inhibitor 4-bromophenacyl bromide (4-BPB), the general COX inhibitor ibuprofen (IB), and the highly selective COX-2 inhibitor NS398. Inhibition of cell invasiveness by 4-BPB (1.0 microM), IB (10.0 microM), and NS398 (10.0 microM) was reversed by the addition of prostaglandin E2 (PGE2). PGE2 alone, however, did not stimulate invasiveness, which suggests that its production is necessary for rendering the cells invasive-permissive but not sufficient for inducing invasiveness. In contrast, we found no significant inhibition of invasion of prostate tumor cells treated with esculetin (1.0 microM) or nordihydroguiaretic acid (1.0 microM), which are specific inhibitors of LOX. We also tested the effect of 4-BPB, IB, NS398, and esculetin on the secretion of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs), as key enzymes in the proteolysis of Matrigel during invasion, using gelatin zymograms and Western blots. Cells that received 4-BPB, IB, or NS398, but not esculetin showed a significant reduction in the levels of proMMP-2, MMP-9, and proMMP-9 in the culture medium. DU-145 cells did not secrete TIMP-1, and the drugs did not alter the secretion of TIMP-2. This work highlights the role played by COX in disturbing the balance between MMPs and TIMPs in prostate cancer cells, and it points to the potential use of COX inibitors, especially COX-2 selective inhibitors, in the prevention and therapy of prostate cancer invasion.

Chromium(VI) induces p53-dependent apoptosis in diploid human lung and mouse dermal fibroblasts.

Some forms of hexavalent chromium [Cr(VI)] are known to cause damage to respiratory-tract tissue and DNA and are thought to be human lung carcinogens. In general, Cr(VI) is mutagenic and carcinogenic at doses that also evoke some cell death, and we previously showed that the predominant mode of death is apoptosis. Because p53 has been shown to initiate apoptosis after genotoxic insults, the objective of these experiments was to determine whether p53 is activated in and necessary for apoptosis of normal diploid human lung fibroblasts (HLF cells) after chromium exposure. By using annexin(V) staining and fluorescent microscopy, we found that Cr(VI) caused up to 14% of HLF cells to undergo apoptosis within 24 h after exposure. In addition, by using western blotting, we found that p53 protein levels increased fourfold to sixfold after exposure to sodium chromate. Because the major function of p53 is as a transcription factor, it must be translocated from the cytoplasm to the nucleus after chromate exposure to be active. Immunofluorescence studies using an antibody against p53 showed that, after chromate exposure, p53 was located in the nucleus of the treated HLF cells. The necessity of p53 for chromium-induced apoptosis was examined in two ways. One approach used dermal fibroblasts from p53 wild-type, heterozygous, and null mice, and the other approach used HLF cells that were transiently transfected with the human papilloma virus E6 gene, which targets p53 for degradation and creates a functional p53-null cell. These studies showed that chromium-induced apoptosis was p53 dependent. Mol. Carcinog. 28:111-118, 2000.

Apoptosis and P53 induction in human lung fibroblasts exposed to chromium (VI): effect of ascorbate and tocopherol.

Some forms of hexavalent chromium [Cr(VI)] are known to cause damage to respiratory tract tissue, and are thought to be human lung carcinogens. Because Cr(VI) is mutagenic and carcinogenic at doses that evoke cell toxicity, the objective of these experiments was to examine the effect of Cr(VI) on the growth, survival, and mode of cell death in normal human lung fibroblasts (HLF cells). DNA adduct formation was monitored as a marker for bioavailability of genotoxic chromium. We also examined the modulation of these endpoints by vitamins C and E. Long-term Cr(VI) exposures were employed, which decreased clonogenic cell survival by 25% to 95% in a dose-dependent manner. The predominant cellular response to Cr(VI) was growth arrest. We found that Cr(VI) caused up to 20% of HLF cells to undergo apoptosis, and documented apoptotic morphology and the phagocytosis of apoptotic bodies by neighboring cells. P53 levels increased 4- to 6-fold in chromium-treated cells. In contrast with previous studies using CHO cells, the present study using HLFs found that pretreatment with either vitamin C or E did not exhibit a significant effect on Cr-induced apoptosis or clonogenic survival. In addition, pretreatment with vitamin C did not affect the p53 induction observed after chromium treatment. Neither vitamin had any effect on Cr-DNA adduct formation. These data indicate that although pretreatment with vitamin C or E alters the spectrum of cellular and/or genetic lesions induced by chromium(VI), neither vitamin altered the initiation or progression of apoptosis in diploid human lung cells.

Overexpression of the glucose-regulated stress gene GRP78 in malignant but not benign human breast lesions.

The 78 kDa glucose-regulated stress protein GRP78 is induced by physiological stress conditions such as hypoxia, low pH, and glucose deprivation which often exist in the microenvironments of solid tumors. Activation of this stress pathway occurs in response to several pro-apoptotic stimuli. In vitro studies have demonstrated a correlation between induced expression of GRP78 and resistance to apoptotic death induced by topoisomerase II-directed drugs. We were interested in characterizing this protein in human breast lesions for potential implications in chemotherapeutic intervention. Surgical specimens of human breast lesions and paired normal tissues from the same patients were flash frozen for these studies. Total RNA and/or protein were extracted from these tissues and used in northern and/or western blot analyses, respectively, to quantify the relative expression of GRP78. Northern blot analysis indicated that 0/5 benign breast lesions, 3/5 estrogen receptor positive (ER+) breast tumors, and 6/9 estrogen receptor negative (ER-) breast tumors exhibited overexpression of GRP78 mRNA compared to paired normal tissues, with fold overexpressions ranging from 1.8 to 20. Western blot analyses correlated with these findings since 0/5 benign breast lesions, 4/6 ER+ breast tumors, and 3/3 ER- breast tumors overexpressed GRP78 protein with fold overexpressions ranging from 1.8 to 19. Immunohistochemical analysis of these tissues demonstrated that the expression of GRP78 was heterogeneous among the cells comprising different normal and malignant glands, but confirmed the overexpression of GRP78 in most of the more aggressive ER- tumors. These results suggest that some breast tumors exhibit adverse microenvironment conditions that induce the overexpression of specific stress genes that may play a role in resistance to apoptosis and decreased chemotherapeutic efficacy.

Prostaglandin A1 inhibits stress-induced NF-kappaB activation and reverses resistance to topoisomerase II inhibitors.

Stress conditions associated with solid tumors lead to the formation of heterogeneous tumor cell subpopulations and insensitivity to cancer chemotherapeutics. In this report, we show that EMT6 mouse mammary tumor cells treated with the chemical stress, brefeldin A (BFA), or the physiological stress, hypoxia, develop resistance to the topoisomerase II (topoII) inhibitors teniposide and etoposide. BFA and hypoxia treatment did not alter intracellular drug concentrations, topoll protein levels, or inhibit topoII activity. BFA and hypoxia did cause the activation of the nuclear transcription factor NF-kappaB. We demonstrate that pretreatment with the synthetic cyclopentenone prostaglandin A1 (PGA1) inhibits stress-induced NF-kappaB activation and reverses BFA- and hypoxia-induced resistance. The reversal of BFA-induced resistance can occur when PGA1 is administered either before or several hours after the induction of stress. Taken together, these data support the involvement of NF-kappaB in stress-induced drug resistance, show that pharmacologic inhibitors of NF-kappaB can disrupt the biological consequences of stress, and imply that inhibitors of NF-kappaB may be useful agents to enhance the clinical efficacy of topoII-directed chemotherapeutics.

Internalization of carcinogenic lead chromate particles by cultured normal human lung epithelial cells: formation of intracellular lead-inclusion bodies and induction of apoptosis.

Occupational exposure to certain particulate hexavalent chromium [Cr(VI)] compounds, such as lead chromate, has been associated with lung cancer and respiratory tract toxicity. We have previously shown that apoptosis is a major mode of death in cultured rodent cells treated with soluble sodium chromate and particulate lead chromate. Here we report the cellular and molecular effects of lead chromate and sodium chromate in normal human lung small airway epithelial (HSAE) cells, which may be one of the targets for Cr(VI)-induced lung cancer and respiratory tract toxicity. Phagocytosed lead chromate particles and intracellular lead-inclusion bodies (LIB) were observed by transmission electron microscopy and confirmed by X-ray analysis. HSAE cells exposed to lead chromate and sodium chromate underwent dose-dependent apoptosis. The cellular uptake and genomic interactions of both Cr and lead (Pb) were examined by inductively coupled plasma mass spectrometry (ICPMS) coupled with a novel, direct-injection high-efficiency nebulizer (DIHEN). Using this approach, we have quantitated a dose-dependent formation of Cr-DNA adducts and DNA-associated Pb in lead chromate-treated HSAE cells. The formation of LIB in normal human lung cells exposed to lead chromate indicates that ionic Pb is released from the particles and thus might contribute to the cell toxicity caused by lead chromate. Internalization and dissolution of lead chromate particles and the interaction of ionic Cr and Pb with DNA, may be components of the mechanism of lead chromate carcinogenesis. Lead chromate-induced apoptosis may be a mechanism to eliminate cells with chromium- and/or lead-damaged DNA.

Differential sensitivity of chromium-mediated DNA interstrand crosslinks and DNA-protein crosslinks to disruption by alkali and EDTA.

Some compounds of hexavalent chromium are well-established carcinogens. Chromium enters mammalian cells in the hexavalent form and is reduced to chromium (III). Treatment of purified DNA with chromium (III) produces DNA-DNA interstrand crosslinks (DDC) which obstruct the progression of DNA polymerases in vitro. DDC were also detected in chromate-treated cultured normal human lung cells using the renaturing agarose gel electrophoresis (RAGE) assay and correlated with base-specific inhibition of DNA replication. Curiously, DDC have gone undetected in studies of cultured cells using the alkaline elution (AE) technique, whereas chromium-mediated DNA-protein crosslinks (DPC) were readily detected by AE. We tested the hypothesis that AE conditions [60 mM tetraethyl ammonium hydroxide (TEA), 20 mM EDTA, pH 12.6, for 16 h at room temperature] dissociate DDC but not DPC using chromium(III)-treated plasmid DNA and the RAGE assay. Dose-dependent chromium-induced DDC were unaffected by TEA (pH 11.8) alone or by more rigorous alkaline denaturation conditions (200 mM NaOH, pH 13.5, for 16 h). DDC were, however, completely disrupted by EDTA (pH 12.6) alone or the combination of TEA and EDTA (pH 12.6). In contrast, DPC remained largely intact under these conditions. Therefore, past AE-based studies which have failed to detect chromium-induced DDC do not prove the absence of this lesion. AE may not be suitable for detecting DDC induced by EDTA-chelatable agents such as metals.

Arrest of replication by mammalian DNA polymerases alpha and beta caused by chromium-DNA lesions.

We have previously shown that trivalent chromium, and hexavalent chromium in the presence of one of its primary in vivo reductants, ascorbate, can bind to DNA and form interstrand crosslinks capable of obstructing replication. This effect was demonstrated in vitro by using Sequenase Version 2.0 T7 DNA polymerase; its parent enzyme, the unmodified T7 DNA polymerase; and Escherichia coli polymerase I large (Klenow) fragment; and it was demonstrated ex vivo by using Taq polymerase and DNA from chromium-treated human lung cells as template. This study was performed to determine whether DNA-bound chromium affects mammalian DNA polymerases in the same manner. Two mammalian enzymes, DNA polymerase alpha and DNA polymerase beta, were used. DNA polymerase alpha is a processive enzyme believed to be the primary lagging-stand synthetase, whereas DNA polymerase beta is a non-processive enzyme believed to function in DNA repair by filling single stranded gaps one base at a time. DNA polymerase arrest assays were performed with each of these enzymes to replicate DNA with toxicologically relevant levels of chromium adducts produced by either trivalent chromium or hexavalent chromium and ascorbate. Both enzymes responded to chromium-DNA damage by arresting replication, and the arrests increased in a dose-dependent manner. Furthermore, the guanine-specific pattern of arrests produced when an exonuclease-free preparation of DNA polymerase beta was used corresponded exactly to the arrest patterns produced in vitro by the exonuclease-free enzyme Sequenase and ex vivo by Taq polymerase. These results suggest that replication arrest may be a common response of polymerases to DNA-chromium lesions and provide a plausible mechanism for the inhibition of DNA synthesis and S-phase cell-cycle delay that occurs in mammalian cells treated with genotoxic chromium compounds.

Chromium-induced genotoxicity and apoptosis: relationship to chromium carcinogenesis (review).

The adverse health effects linked with chromium (Cr) exposure, the role of solubility and chemical speciation of Cr compounds, and the diverse cellular and molecular effects of Cr make the study of Cr carcinogenesis and toxicology very interesting and complex. Certain Cr compounds are prominent metal carcinogens in both occupational and environmental settings. Inhaled particulate forms of hexavalent Cr [Cr(VI)] cause lung cancer as well as lung toxicity. Some of the important factors in determining the biological outcome of Cr exposure include the bioavailability, chemical speciation and solubility of Cr compounds, intracellular reduction, and interaction of Cr with DNA. The stable oxidation states of Cr found in nature are Cr(III) and Cr(VI). Cr(III) is unable to enter cells but Cr(VI) enters into cells through membrane anionic transporters. Intracellular Cr(VI) is metabolically reduced to the ultimate Cr(III). Cr(VI) does not react with macromolecules such as DNA, RNA, proteins and lipids. However, both Cr(III) and the reductional intermediate Cr(V) are capable of co-ordinate covalent interactions with macromolecules. At the genomic level, Cr genotoxicity manifests as gene mutations, several types of DNA lesions and inhibition of macromolecular synthesis. At the cellular level, Cr exposure may lead to cell cycle arrest, apoptosis, premature terminal growth arrest, or neoplastic transformation. Cr-induced DNA-DNA interstrand crosslinks (DDC), the tumor suppressor gene p53 and oxidative processes are some of the major factors that may play a significant role in determining the cellular outcome in response to Cr exposure. We have utilized cellular, molecular, pharmacological, and genetic approaches to understand the interrelationship between Cr-induced genotoxicity, apoptosis and carcinogenesis. This review is based on the results and inferences of this research. We hope this review will clarify existing concepts and also introduce novel perspectives in chromium carcinogenesis research.

Prevention of brefeldin A-induced resistance to teniposide by the proteasome inhibitor MG-132: involvement of NF-kappaB activation in drug resistance.

Brefeldin A, an agent that disrupts protein transport from the endoplasmic reticulum to the Golgi, induces the expression of GRP78 and the activation of nuclear factor (NF)-kappaB in cells. Treatment of cells with brefeldin A causes the development of resistance to topoisomerase II-directed agents, such as etoposide and doxorubicin. In this study, we show that treatment of EMT6 mouse mammary tumor cells with brefeldin A strongly induces GRP78 mRNA (8.5-fold) and resistance to teniposide (VM26). Treatment with okadaic acid causes a minor increase in GRP78 mRNA (2.1-fold) yet still induces resistance to VM26 as effectively as brefeldin A. In contrast, cells treated with castanospermine show a moderate increase in GRP78 mRNA (3.9-fold) but no resistance to VM26. These data imply that GRP78 induction does not mediate the development of drug resistance. An alternative mechanism of drug resistance may involve activation of the transcription factor, NF-kappaB, and we show that both brefeldin A and okadaic acid activate NF-kappaB in EMT6 cells. Furthermore, we demonstrate that treatment with the proteasome inhibitor MG-132 blocks the activation of NF-kappaB and prevents the development of resistance to VM26 induced by brefeldin A. Collectively, these results suggest that the resistance to VM26 in EMT6 cells treated with brefeldin A is mediated by the activation of NF-kappaB rather than the induction of GRP78. Our results also suggest that inhibition of NF-kappaB activation in tumor cells may increase the efficacy of topoisomerase II-directed agents in chemotherapy.

Sensitive quantitation of chromium-DNA adducts by inductively coupled plasma mass spectrometry with a direct injection high-efficiency nebulizer.

A novel method is described for the sensitive detection of chromium-DNA adducts. Chromium-DNA adducts were determined in 1 microgram of DNA from normal human lung fibroblasts exposed to sodium chromate using microscale flow injection analysis with a direct injection high-efficiency nebulizer and inductively coupled plasma mass spectrometry detection. The frequency of Cr-DNA adducts increased in a dose-dependent sigmoidal manner, indicating saturation and toxicity. The low detection limits (on the order of parts per trillion) allows the detection of as few as two Cr adducts per 10,000 bases, which, coupled with the small DNA sample requirement, makes this technique suitable for measuring metal-DNA adducts as biomarkers of exposure to toxic and carcinogenic metals such as Cr, in cultured cells, animals, and humans.