Biomedical subjects

J F Hatcher

Publications and source records attributed to J F Hatcher.

At least 19 recordsLinked to original sources

PubMed2007

Secretory phospholipase A2 IIA is up-regulated by TNF-alpha and IL-1alpha/beta after transient focal cerebral ischemia in rat.

Cerebral ischemia initiates an inflammatory response in the brain that is associated with the induction of a variety of cytokines, including tumor necrosis factor-alpha (TNF-alpha) and interleukin-1alpha/beta (IL-1alpha/beta) that contributes to stroke injury. Transient middle cerebral artery occlusion (tMCAO) in spontaneously hypertensive rat (SHR) resulted in significant increases in TNF-alpha and IL-1beta levels. We have previously demonstrated up-regulation of secretory phospholipase A2 IIA (sPLA2 IIA) mRNA and protein expression, increased PLA2 activity, and loss of phosphatidylcholine after 1-h tMCAO and 24-h reperfusion in SHR. Treatment with TNF-alpha antibody or IL-1 receptor antagonist significantly attenuated infarction volume, sPLA2 IIA protein expression, PLA2 activity and significantly restored phosphatidylcholine levels after tMCAO. This suggests that cytokine induction up-regulates sPLA2 IIA protein expression, resulting in altered lipid metabolism that contributes to stroke injury.

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PubMed2006

Lipids and lipidomics in brain injury and diseases.

Lipidomics is systems-level analysis and characterization of lipids and their interacting moieties. The amount of information in the genomic and proteomic fields is greater than that in the lipidomics field, because of the complex nature of lipids and the limitations of tools for analysis. The main innovation during recent years that has spurred advances in lipid analysis has been the development of new mass spectroscopic techniques, particularly the "soft ionization" techniques electrospray ionization and matrix-assisted laser desorption/ionization. Lipid metabolism may be of particular importance for the central nervous system, as it has a high concentration of lipids. The crucial role of lipids in cell signaling and tissue physiology is demonstrated by the many neurological disorders, including bipolar disorders and schizophrenia, and neurodegenerative diseases such as Alzheimer's, Parkinson's, and Niemann-Pick diseases, that involve deregulated lipid metabolism. Altered lipid metabolism is also believed to contribute to cerebral ischemic (stroke) injury. Lipidomics will provide a molecular signature to a certain pathway or a disease condition. Lipidomic analyses (characterizing complex mixtures of lipids and identifying previously unknown changes in lipid metabolism) together with RNA silencing, using small interfering RNA (siRNA), may provide powerful tools to elucidate the specific roles of lipid intermediates in cell signaling and open new opportunities for drug development.

Animals↗

PubMed2005

Phospholipase A2, reactive oxygen species, and lipid peroxidation in cerebral ischemia.

Ischemic stroke is caused by obstruction of blood flow to the brain, resulting in energy failure that initiates a complex series of metabolic events, ultimately causing neuronal death. One such critical metabolic event is the activation of phospholipase A2 (PLA2), resulting in hydrolysis of membrane phospholipids and release of free fatty acids including arachidonic acid, a metabolic precursor for important cell-signaling eicosanoids. PLA2 enzymes have been classified as calcium-dependent cytosolic (cPLA2) and secretory (sPLA2) and calcium-independent (iPLA2) forms. Cardiolipin hydrolysis by mitochondrial sPLA2 disrupts the mitochondrial respiratory chain and increases production of reactive oxygen species (ROS). Oxidative metabolism of arachidonic acid also generates ROS. These two processes contribute to formation of lipid peroxides, which degrade to reactive aldehyde products (malondialdehyde, 4-hydroxynonenal, and acrolein) that covalently bind to proteins/nucleic acids, altering their function and causing cellular damage. Activation of PLA2 in cerebral ischemia has been shown while other studies have separately demonstrated increased lipid peroxidation. To the best of our knowledge no study has directly shown the role of PLA2 in lipid peroxidation in cerebral ischemia. To date, there are very limited data on PLA2 protein by Western blotting after cerebral ischemia, though some immunohistochemical studies (for cPLA2 and sPLA2) have been reported. Dissecting the contribution of PLA2 to lipid peroxidation in cerebral ischemia is challenging due to multiple forms of PLA2, cardiolipin hydrolysis, diverse sources of ROS arising from arachidonic acid metabolism, catecholamine autoxidation, xanthine oxidase activity, mitochondrial dysfunction, activated neutrophils coupled with NADPH oxidase activity, and lack of specific inhibitors. Although increased activity and expression of various PLA2 isoforms have been demonstrated in stroke, more studies are needed to clarify the cellular origin and localization of these isoforms in the brain, their responses in cerebral ischemic injury, and their role in oxidative stress.

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PubMed2005

CDP-choline liposomes provide significant reduction in infarction over free CDP-choline in stroke.

Cytidine-5'-diphosphocholine (CDP-choline, Citicoline, Somazina) is in clinical use (intravenous administration) for stroke treatment in Europe and Japan, while USA phase III stroke clinical trials (oral administration) were disappointing. Others showed that CDP-choline liposomes significantly increased brain uptake over the free drug in cerebral ischemia models. Liposomes were formulated as DPPC, DPPS, cholesterol, GM(1) ganglioside; 7/4/7/1.57 molar ratio or 35.8/20.4/35.8/8.0 mol%. GM(1) ganglioside confers long-circulating properties to the liposomes by suppressing phagocytosis. CDP-choline liposomes deliver the agent intact to the brain, circumventing the rate-limiting, cytidine triphosphate:phosphocholine cytidylyltransferase in phosphatidylcholine synthesis. Our data show that CDP-choline liposomes significantly ( P < 0.01) decreased cerebral infarction (by 62%) compared to the equivalent dose of free CDP-choline (by 26%) after 1 h focal cerebral ischemia and 24 h reperfusion in spontaneously hypertensive rats. Beneficial effects of CDP-choline liposomes in stroke may derive from a synergistic effect between the phospholipid components of the liposomes and the encapsulated CDP-choline.

Animals↗

PubMed2005

Cytidine 5'-diphosphocholine (CDP-choline) in stroke and other CNS disorders.

Brain phosphatidylcholine (PC) levels are regulated by a balance between synthesis and hydrolysis. Pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 (IL-1alpha/beta) activate phospholipase A(2) (PLA(2)) and PC-phospholipase C (PC-PLC) to hydrolyze PC. PC hydrolysis by PLA(2) releases free fatty acids including arachidonic acid, and lyso-PC, an inhibitor of CTP-phosphocholine cytidylyltransferase (CCT). Arachidonic acid metabolism by cyclooxygenases/lipoxygenases is a significant source of reactive oxygen species. CDP-choline might increase the PC levels by attenuating PLA(2) stimulation and loss of CCT activity. TNF-alpha also stimulates proteolysis of CCT. TNF-alpha and IL-1beta are induced in brain ischemia and may disrupt PC homeostasis by increasing its hydrolysis (increase PLA(2) and PC-PLC activities) and inhibiting its synthesis (decrease CCT activity). The beneficial effects of CDP-choline may result by counteracting TNF-alpha and/or IL-1 mediated events, integrating cytokine biology and lipid metabolism. Re-evaluation of CDP-choline phase III stroke clinical trial data is encouraging and future trails are warranted. CDP-choline is non-xenobiotic, safe, well tolerated, and can be considered as one of the agents in multi-drug treatment of stroke.

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PubMed2002

Citicoline mechanisms and clinical efficacy in cerebral ischemia.

Citicoline, an intermediate in the biosynthesis of phosphatidylcholine (PtdCho), has shown beneficial effects in various CNS injury models and neurodegenerative diseases. PtdCho hydrolysis by phospholipase A(2) (PLA(2)) after cerebral ischemia and reperfusion yields arachidonic acid (ArAc) and lyso-PtdCho. ArAc oxidative metabolism results in formation of reactive oxygen species and lipid peroxides. Lyso-PtdCho could inhibit activity of cytidine triphosphate-phosphocholine cytidylyltransferase (the rate-limiting enzyme in PtdCho biosynthesis), resulting in impaired PtdCho synthesis. Citicoline significantly increased glutathione levels and attenuated release of ArAc and the loss of PtdCho, cardiolipin, and sphingomyelin following transient cerebral ischemia. These effects could be explained by an effect of citicoline on PLA(2). Based on these observations, a mechanism has been hypothesized. This Mini-Review summarizes recent experimental data on the effects of citicoline in cerebral ischemia and evaluates several factors that might have hindered efficacy of citicoline in stroke clinical trials in the United States. Clinical stroke trials of citicoline in Europe and Japan have demonstrated beneficial effects. U.S. trials shown only marginal effects, which might be due to the 24 hr time window, the dose and route of administration, and the stringency of the primary outcome parameters. Recent evaluation of U.S. clinical data suggests that reduction of infarct growth may be a more sensitive measure of the citicoline effect than improvement on the NIH Stroke Scale (NIHSS) by > or =7 points. The citicoline neuroprotective mechanism has not been clearly identified, and its potential in stroke treatment might still be fully recognized in the United States. The clinical efficacy of citicoline should be examined further in light of the recent phase III stroke clinical trials and experimental data for cerebral ischemia.

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PubMed2002

Loss of cardiolipin and mitochondria during programmed neuronal death: evidence of a role for lipid peroxidation and autophagy.

Cardiolipin, a lipid of the mitochondrial inner membrane, is lost from many types of cells during apoptotic death. Here we show that the cardiolipin content of nerve growth factor (NGF)-deprived rat sympathetic neurons undergoing apoptotic death in cell culture decreased before extensive loss of mitochondria from the cells. By 18-24 h after NGF deprivation, many neurons did not stain with the cardiolipin-specific dye, Nonyl Acridine Orange, suggesting complete loss of cardiolipin. Gas chromatography confirmed the decline of cardiolipin content in NGF-deprived neurons. Electron microscopy and immunoblots for the mitochondrial-specific protein, heat shock protein 60 (HSP60), revealed that there was only a slight decrease in mitochondrial mass at this time. Cardiolipin loss after NGF deprivation was concurrent with increased production of mitochondrial-derived reactive oxygen species [Kirkland, R.A., Franklin, J.L., 2001. J. Neurosci. 21, 1949-1963] and increased lipid peroxidation. Compounds having antioxidant effects blocked peroxidation, loss of cardiolipin, and the decrease of mitochondrial mass in NGF-deprived neurons. These compounds also blocked an increase in the number of lysosomes and autophagosomes in NGF-deprived cells. The findings reported here show that the important mitochondrial inner membrane lipid, cardiolipin, is lost from mitochondria during neuronal apoptosis and that this loss occurs before significant loss of mitochondria from cells. They suggest that the loss of cardiolipin is mediated by free radical oxygen.

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PubMed2002

Citicoline: neuroprotective mechanisms in cerebral ischemia.

Cytidine-5'-diphosphocholine (citicoline or CDP-choline), an intermediate in the biosynthesis of phosphatidylcholine (PtdCho), has shown beneficial effects in a number of CNS injury models and pathological conditions of the brain. Citicoline improved the outcome in several phase-III clinical trials of stroke, but provided inconclusive results in recent clinical trials. The therapeutic action of citicoline is thought to be caused by stimulation of PtdCho synthesis in the injured brain, although the experimental evidence for this is limited. This review attempts to shed some light on the properties of citicoline that are responsible for its effectiveness. Our studies in transient cerebral ischemia suggest that citicoline might enhance reconstruction (synthesis) of PtdCho and sphingomyelin, but could act by inhibiting the destructive processes (activation of phospholipases). Citicoline neuroprotection may include: (i) preserving cardiolipin (an exclusive inner mitochondrial membrane component) and sphingomyelin; (ii) preserving the arachidonic acid content of PtdCho and phosphatidylethanolamine; (iii) partially restoring PtdCho levels; (iv) stimulating glutathione synthesis and glutathione reductase activity; (v) attenuating lipid peroxidation; and (vi) restoring Na(+)/K(+)-ATPase activity. These observed effects of citicoline could be explained by the attenuation of phospholipase A(2) activation. Based on these findings, a singular unifying mechanism has been hypothesized. Citicoline also provides choline for synthesis of neurotransmitter acetylcholine, stimulation of tyrosine hydroxylase activity and dopamine release.

Animals↗

PubMed2001

Does CDP-choline modulate phospholipase activities after transient forebrain ischemia?

Ten min forebrain ischemia/1-day reperfusion resulted in significant decreases in total phosphatidylcholine (PtdCho), phosphatidylinositol (PtdIns), and cardiolipin in gerbil hippocampus. CDP-choline restored cardiolipin levels, arachidonic acid content of PtdCho, partially but significantly restored total PtdCho, and had no effect on PtdIns. These data suggest that CDP-choline prevented the activation of phospholipase A(2) (rather than inhibiting phospholipase A(2) activity) but did not affect activities of PtdCho-phospholipases C and/or D, or phosphoinositide-phospholipase C. CDP-choline also provided significant protection for hippocampal CA(1) neurons.

Animals↗

PubMed2001

Effects of citicoline on phospholipid and glutathione levels in transient cerebral ischemia.

BACKGROUND AND PURPOSE: Cytidine-5'-diphosphocholine (citicoline or CDP-choline) is an essential intermediate in the biosynthesis of phosphatidylcholine, an important component of the neural cell membrane. Citicoline provided significant neuroprotection after transient forebrain ischemia in gerbils. This study was undertaken to examine changes and effects of citicoline on phospholipids and glutathione synthesis after transient cerebral ischemia and reperfusion. METHODS: Ten-minute transient forebrain ischemia was induced by bilateral carotid artery occlusion in male Mongolian gerbils with reperfusion up to 6 days. Citicoline (500 mg/kg IP in saline) was given to gerbils just after the end of ischemia, at 3-hour reperfusion, and daily thereafter until 1 day before euthanasia. Hippocampal lipids were extracted and analyzed by thin-layer and gas chromatography. Glutathione was measured by using an enzymatic recycling assay. Glutathione reductase activity was determined by measuring NADPH oxidation. RESULTS: Significant decreases in phospholipids occurred at 1-day reperfusion. Citicoline significantly restored the phosphatidylcholine, sphingomyelin, and cardiolipin levels but did not affect phosphatidylinositol and phosphatidylserine at 1 day. The phospholipids returned to sham levels over days 2 to 6 and were unaffected by citicoline. Ceramide levels significantly increased by 3 and 6 days of reperfusion and were unaltered by citicoline. Ischemia resulted in significant decreases in glutathione and glutathione reductase activity over 3 days of reperfusion. Citicoline significantly increased total glutathione and glutathione reductase activity and decreased the glutathione oxidation ratio, an indicator of glutathione redox status. CONCLUSIONS: Our data indicated that the effects of citicoline on phospholipids occurred primarily during the first day of reperfusion, with effects on glutathione being important over the 3-day reperfusion period.

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PubMed2000

Neuroprotection by group I metabotropic glutamate receptor antagonists in forebrain ischemia of gerbil.

Stimulation of group I metabotropic glutamate receptors (mGluR 1 and 5) activates G-protein coupled-phospholipase C (PLC) to release 1,2-diacylglycerol (DAG) and arachidonic acid (ArAc). To elucidate the role of group I mGluR, we tested the effects of (S)-alpha-methyl-4-carboxy-phenylglycine (MCPG, mGluR 1 and 5 antagonist), 1-aminoindan-1,5-dicarboxylic acid (AIDA, mGluR 1a specific antagonist) and 2-methyl-6-(phenylethynyl) pyridine (MPEP, mGluR 5 antagonist) on ArAc release and neuronal survival after transient forebrain ischemia in gerbils. Ischemia resulted in (a) significant release of ArAc at 1-day reperfusion and (b) significant neuronal death in the hippocampal CA1 subfield after 6-day reperfusion. MCPG and MPEP decreased ArAc release and also significantly increased neuronal survival. AIDA was less effective in decreasing ArAc release and had no effect on neuronal death. These results suggest that activation of mGluR 5 may be an important pathway in ArAc release and neuronal death after transient ischemia.

Animals↗

PubMed2000

Lipid alterations in transient forebrain ischemia: possible new mechanisms of CDP-choline neuroprotection.

We have previously demonstrated that cytidine 5'-diphosphocholine (CDP-choline or citicoline) attenuated arachidonic acid (ArAc) release and provided significant protection for the vulnerable hippocampal CA(1) neurons of the cornu ammonis after transient forebrain ischemia of gerbil. ArAc is released by the activation of phospholipases and the alteration of phosphatidylcholine (PtdCho) synthesis. Released ArAc is metabolized by cyclooxygenases/lipoxygenases to form eicosanoids and reactive oxygen species (ROS). ROS contribute to neurotoxicity through generation of lipid peroxides and the cytotoxic byproducts 4-hydroxynonenal and acrolein. ArAc can also stimulate sphingomyelinase to produce ceramide, a potent pro-apoptotic agent. In the present study, we examined the changes and effect of CDP-choline on ceramide and phospholipids including PtdCho, phosphatidylethanolamine (PtdEtn), phosphatidylinositol (PtdIns), phosphatidylserine (PtdSer), sphingomyelin, and cardiolipin (an exclusive inner mitochondrial membrane lipid essential for electron transport) following ischemia/1-day reperfusion. Our studies indicated significant decreases in total PtdCho, PtdIns, PtdSer, sphingomyelin, and cardiolipin and loss of ArAc from PtdEtn in gerbil hippocampus after 10-min forebrain ischemia/1-day reperfusion. CDP-choline (500 mg/kg i.p. immediately after ischemia and at 3-h reperfusion) significantly restored the PtdCho, sphingomyelin, and cardiolipin levels as well as the ArAc content of PtdCho and PtdEtn but did not affect PtdIns and PtdSer. These data suggest multiple beneficial effects of CDP-choline: (1) stabilizing the cell membrane by restoring PtdCho and sphingomyelin (prominent components of outer cell membrane), (2) attenuating the release of ArAc and limiting its oxidative metabolism, and (3) restoring cardiolipin levels.

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PubMed2000

Elevated N1-acetylspermidine levels in gerbil and rat brains after CNS injury.

The polyamine system is very sensitive to different pathological states of the brain and is perturbed after CNS injury. The main modifications are significant increases in ornithine decarboxylase activity and an increase in tissue putrescine levels. Previously we have shown that the specific polyamine oxidase (PAO) inhibitor N1,N4-bis(2,3-butadienyl)-1,4-butanediamine (MDL 72527) reduced the tissue putrescine levels, edema, and infarct volume after transient focal cerebral ischemia in spontaneously hypertensive rats and traumatic brain injury of Sprague-Dawley rats. In the present study, N1-acetyl-spermidine accumulation was greater in injured brain regions compared with sham or contralateral regions following inhibition of PAO by MDL 72527. This indicates spermidine/spermine-N1-acetyltransferase (SSAT) activation after CNS injury. The observed increase in N1-acetylspermidine levels at 1 day after CNS trauma paralleled the decrease in putrescine levels after treatment with MDL 72527. This suggests that the increased putrescine formation at 1 day after CNS injury is mediated by the SSAT/PAO pathway, consistent with increased SSAT mRNA after transient ischemia.

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PubMed1999

CDP-choline: neuroprotection in transient forebrain ischemia of gerbils.

CDP-choline is a rate-limiting intermediate in the biosynthesis of phosphatidylcholine (PtdCho), an important component of the neural cell membrane. The ability of CDP-choline to alter phospholipid metabolism is an important function in the treatment of ischemic injury. Exogenous treatment with CDP-choline stimulates PtdCho synthesis and prevents release of free fatty acids (FFA), especially arachidonic acid (AA), after ischemia/reperfusion. Phase III clinical trials of CDP-choline in the treatment of stroke are currently underway. Here we report the neuroprotection by CDP-choline in transient forebrain ischemia of gerbils. CDP-choline significantly attenuated the blood-brain barrier (BBB) dysfunction after ischemia with 6-hr reperfusion, and considerably reduced the increase of AA in FFA and leukotriene C(4) (LTC(4)) synthesis at 1 day. Edema was significantly elevated after 1 and 2 days, but attained maximum at 3-day reperfusion. CDP-choline substantially attenuated edema at 3 days. Ischemia resulted in 80 +/- 8% CA(1) hippocampal neuronal death after 6-day reperfusion, and CDP-choline provided 65 +/- 6% neuroprotection. CDP-choline may act by increasing PtdCho synthesis via two pathways: (1) conversion of 1, 2-diacylglycerol to PtdCho, and (2) biosynthesis of S-adenosyl-L-methionine, thus stabilizing the membrane and reducing AA release and metabolism to leukotriene C(4). This would result in decreased toxicity due to AA, leukotrienes, oxygen radicals, lipid peroxidation, and altered glutamate uptake, thus limiting BBB dysfunction, edema and providing neuroprotection.

Animals↗

PubMed1999

Arachidonic acid and leukotriene C4: role in transient cerebral ischemia of gerbils.

Accumulation of arachidonic acid (AA) is greatest in brain regions most sensitive to transient ischemia. Free AA released after ischemia is either: 1) reincorporated into the membrane phospholipids, or 2) oxidized during reperfusion by lipoxygenases and cyclooxygenases, producing leukotrienes (LT), prostaglandins, thromboxanes and oxygen radicals. AA, its metabolite LTC4 and lipid peroxides (generated during AA metabolism) have been implicated in the blood-brain barrier (BBB) dysfunction, edema and neuronal death after ischemia/reperfusion. This report describes the time course of AA release, LTC4 accumulation and association with the physiological outcome during transient cerebral ischemia of gerbils. Significant amount of AA was detected immediately after 10 min ischemia (0 min reperfusion) which returned to sham levels within 30 min reperfusion. A later release of AA occurred after 1 d. LTC4 levels were elevated at 0-6 h and 1 d after ischemia. Increased lipid peroxidation due to AA metabolism was observed between 2-6 h. BBB dysfunction occurred at 6 h. Significant edema developed at 1 and 2 d after ischemia and reached maximum at 3 d. Ischemia resulted in approximately 80% neuronal death in the CA1 hippocampal region. Pretreatment with a 5-lipoxygenase inhibitor, AA861 resulted in significant attenuation of LTC4 levels (Baskaya et al. 1996. J. Neurosurg. 85: 112-116) and CA1 neuronal death. Accumulation of AA and LTC4, together with highly reactive oxygen radicals and lipid peroxides, may alter membrane permeability, resulting in BBB dysfunction, edema and ultimately to neuronal death.

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PubMed1998

Simultaneous assay of ornithine decarboxylase and polyamines after central nervous system injury in gerbil and rat.

Ornithine decarboxylase (ODC) is considered the rate-limiting enzyme in polyamine biosynthesis. An increase in putrescine (a natural polyamine) synthesis after central nervous system (CNS) injury appears to be involved in blood-brain barrier dysfunction, development of vasogenic edema and neuronal death. An improved method is described to determine the ODC activity as well as polyamine levels from the same brain tissue. The polyamine results showed no significant differences from data obtained with the conventional assay. The advantages of this method are to: (1) minimize the number of animals needed for the study, and (2) eliminate any internal inconsistencies resulting from use of two independent groups of animals for ODC and polyamine measurements. Using this method, ODC activities and polyamine levels were measured in cortices and hippocampi from global transient ischemia of gerbils and traumatic brain injury (TBI) of rats.

Animals↗

PubMed1998

Fluorometric assay of nitrite and nitrate in brain tissue after traumatic brain injury and cerebral ischemia.

Nitric oxide synthase (NOS) is distributed within the brain, and nitric oxide (NO) is felt to be involved in the pathophysiology of deterioration after head injury and cerebral ischemia. This study determined the levels of the stable end products of NOS (NOx=nitrite+nitrate) after traumatic brain injury (TBI) and transient cerebral ischemia. A fluorometric assay using nitrate reductase and the NADPH regenerating system was used to quantitate NOx in ultrafiltered (10-kDa cutoff) cortical and hippocampal extracts after reduction of nitrate. In TBI rats, both the plasma and tissue showed a sharp increase in NOx levels 5 min after injury. Plasma NOx returned to control levels by 2 h after injury. Ipsilateral-cortex NOx levels returned to control levels approximately 6 h after injury and remained constant from 6-24 h. Contralateral-cortex returned near to control levels after 1 h. Hippocampus also followed a similar trend. In gerbils, there was a significant elevation in tissue NOx levels immediately after 10 min transient cerebral ischemia, which gradually returned to control levels over 24 h reperfusion. This striking burst of NO synthesis immediately after injury is clearly evident whether the injury is head trauma or ischemia, or whether the measurements were performed on tissue or plasma. It is unknown whether endothelial NOS, neuronal NOS, or both caused the elevation of the NO end products seen after the CNS insults.

2-Naphthylamine↗

PubMed1996

Neoplastic transformation and DNA-binding of 4,4'-methylenebis(2-chloroaniline) in SV40-immortalized human uroepithelial cell lines.

The tumorigenic transformation of certain occupationally significant chemicals, such as N-hydroxy-4-4'-methylenebis[2-chloroaniline] (N-OH-MOCA), N-hydroxy-ortho-toluidine (N-OH-OT), 2-phenyl-1,4-benzoquinone (PBQ) and N-hydroxy-4-aminobiphenyl (N-OH-ABP) were tested in vitro using the well established SV40-immortalized human uroepithelial cell line SV-HUC.PC. SV-HUC cells were exposed in vitro to varying concentrations of N-OH-MOCA, N-OH-OT, N-OH-ABP and PBQ that caused approximately 25% and 75% cytotoxicity. The carcinogen treated cells were propagated in culture for about six weeks and subsequently injected subcutaneously into athymic nude mice. Two of the fourteen different groups of SV-HUC.PC treated with different concentrations of N-OH-MOCA, and one of the three groups exposed to N-OH-ABP, formed carcinomas in athymic nude mice. 32P-postlabeling analyses of DNA isolated from SV-HUC.PC after exposure to N-OH-MOCA revealed one major and one minor adduct. The major adduct has been identified as the N-(deoxyadenosin-3',5'-bisphospho-8-yl)-4-amino-3-chlorob enz yl alcohol (pdAp-ACBA) and the minor adduct as N-(deoxyadenosin-3',5'-bisphospho-8-yl)-4-amino-3-chlorot oluene (pdApACT). Furthermore, SV-HUC.PC cytosols catalyzed the binding of N-OH-MOCA to DNA, in the presence of acetyl-CoA, to yield similar adducts. The same adducts were also formed by chemical interaction of N-OH-MOCA with calf thymus DNA, suggesting that the aryl nitrenium ion may be the ultimate reactive species responsible for DNA binding. The tumorigenic activity of N-OH-MOCA in this highly relevant in vitro transformation model, coupled with the findings that SV-HUC.PC cells formed DNA-adducts in vitro and contained enzyme systems that activated N-OH-MOCA to reactive electrophilic species that bound to DNA, strongly suggest that MOCA could be a human bladder carcinogen. These findings are consistent with the International Agency for Research on Cancer's classification of MOCA as a probable human carcinogen.

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