Nitric oxide synthase activity in the hippocampus,frontal cerebral cortex,and cerebellum of the guinea pig: Ontogeny and in vitro ethanol exposure

Decreased nitric oxide (NO) formation, resulting from inhibition of NO synthase (NOS), may be important in the pathogenesis of ethanol central nervous system teratogenesis. The objectives of this study were to determine the ontogeny of NOS activity in the hippocampus, frontal cerebral cortex, and cerebellum of the developing guinea pig, and to test the hypothesis that direct exposure to ethanol inhibits NOS activity in these brain regions at selected developmental ages. NOS activity was quantitated by an optimized radiometric assay. The ontogeny study demonstrated that NOS activity in the hippocampus and frontal cortex was not fully developed prenatally, and apparently increased during postnatal life to attain adult level of activity at postnatal day > 60. In the cerebellum, NOS activity increased during prenatal life to an apparent maximum in the mature near-term fetus at gestational day 63 (term, about 68 days), and then apparently declined during postnatal life to attain adult level of activity. In vitro ethanol exposure (25–100 mM) did not affect NOS activity in the hippocampus, frontal cortex, or cerebellum at any developmental age studied. These data indicate that, although the ontogeny of NOS activity varies between brain regions, ethanol does not directly affect NOS activity in the developing guinea pig. The effects of acute and chronic in utero ethanol exposure on NOS activity in these brain regions are currently being investigated.     

Chronic ethanol ingestion increases nitric oxide production in the lung.

Chronic ethanol (EtOH) ingestion increases the incidence of acute respiratory distress syndrome. The mechanisms underlying EtOH-induced susceptibility to lung injury continue to be defined. This study examines the hypothesis that EtOH increases endothelial nitric oxide synthase (eNOS) expression and activity in the lungs of a rat model of chronic EtOH ingestion. Male Sprague-Dawley rats were fed liquid diets containing EtOH (36% of calories) or maltose-dextrin as an isocaloric substitution for EtOH (control) for 6 weeks. Selected animals were also treated with the angiotensin-converting enzyme (ACE) inhibitor lisinopril (3 mg/l in diet) for 6 weeks. At study completion, animals were sacrificed, and lung tissue was collected for assays of nitric oxide (NO) metabolism or pulmonary microvascular endothelial cells (MVEC) were isolated for analysis of NO release. Compared to the control diet, chronic EtOH ingestion increased lung H2O2 production, eNOS expression and activity, lung cyclic guanosine monophosphate (cGMP) content, and levels of protein nitration and oxidation. MVEC from animals with chronic EtOH ingestion released greater amounts of NO. EtOH-induced increases in lung H2O2 production, eNOS expression and activity, cGMP content, protein nitration and oxidation, and MVEC NO production were all attenuated by treatment with lisinopril. Chronic EtOH ingestion stimulates ACE-dependent increases in NO production in the lung. These novel findings indicate that chronic EtOH ingestion increases reactive species production in the lung parenchyma and provide new insights into mechanisms by which EtOH causes phenotypic alterations in the lung and alters the lung's response to inflammatory stimuli.     

亚氨乙基赖氨酸拮抗噪声损伤豚鼠听力的实验研究

目的　探讨亚氨乙基赖氨酸对豚鼠噪声性损伤的拮抗作用。方法　选健康白色红目豚鼠 4 0只 ,随机分为 4组 :A组为正常对照组 ,B组为噪声组 ,C组为噪声 +药物组 ,D组为亚氨乙基赖氨酸组。B、C组豚鼠暴露于 115dB白噪声连续 6d ,每天连续 6h ;C组豚鼠每天腹腔注射亚氨乙基赖氨酸 10mg/kg ,B组豚鼠腹腔注射等量的生理盐水 ,D组豚鼠每天腹腔注射亚氨乙基赖氨酸 10mg/kg,并不暴露于噪声。各组豚鼠在实验前、后均行ABR听阈检测。用免疫组化方法检测诱生型一氧化氮合成酶 (NOSⅡ )在各组豚鼠耳蜗的表达。各组豚鼠耳蜗行扫描电镜检查。观察比较各组豚鼠ABR听阈、NOSⅡ染色强弱及耳蜗形态。结果　实验前 ,A、B、C、D各组间ABR听阈的差异无显著性 (P >0 .0 5 )。实验后 ,A组、D组ABR听阈无明显改变 ,而B组和C组ABR听阈则有明显改变 ,B组ABR听阈为 (6 0 .2 3± 11.2 3)dB ,C组为 (38.4 6± 7.2 4 )dB ,两组比较 ,差异有显著性 (P <0 .0 5 )。NOSⅡ在A、D组耳蜗表达阴性 ,B组耳蜗表达较强 ,C组耳蜗表达较弱。B组豚鼠耳蜗外毛细胞损伤较C组重。结论　NOSⅡ在噪声所致豚鼠耳蜗损伤中呈阳性表达 ,亚氨乙基赖氨酸能抑制NOSⅡ的活力 ,且对噪声所致豚鼠耳蜗损伤有拮抗作用 ,表明一氧化氮在噪声性聋的发病中起重要作用。     

锌对急性缺氧小鼠大脑皮层NOS活力和nNOS蛋白表达的影响

目的观察锌对急性缺氧小鼠大脑皮层一氧化氮合酶 (NOS)和神经元型一氧化氮合酶 (nNOS)阳性神经元的变化 ,探讨锌抗脑缺氧的作用机制。方法制备小鼠急性缺氧模型 ,采用NADPH -d组织化学和nNOS免疫组织化学方法 ,研究给锌组和不给锌组急性缺氧小鼠大脑皮层NOS和nNOS阳性神经元数量的变化。结果给锌组比不给锌组小鼠的耐缺氧时间延长 33.0 6 %(P <0 .0 5 ) ,大脑皮层NOS和nNOS阳性神经元数量分别减少 18.0 3%和 2 1.6 3% ,(P <0 .0 5 )。结论急性缺氧时锌通过减少皮层NOS活力和nNOS水平而发挥其抗脑缺氧作用。     

Leishmania donovani: identification of stimulatory soluble antigenic proteins using cured human and hamster lymphocytes for their prophylactic potential against visceral leishmaniasis

Most of the studies for the identification of prophylactic antigens that elicit T cell responses were concentrated on membrane proteins of Leishmania donovani. This study was taken up to assess L. donovani soluble promastigote antigens for their ability to stimulate proliferation of peripheral blood mononuclear cells (PBMCs) from cured visceral leishmaniasis (VL) patients, endemic and non-endemic controls and lymphocytes/peritoneal macrophages of cured hamsters. The soluble protein was subjected to sequential precipitation with saturated ammonium sulphate (20%, 40%, 60% and 80%), of which largely 80% fractioned protein showed significant cellular responses in cured patients and hamsters. This fraction was further fractionated into five sub fractions by preparative SDS-PAGE and subjected to re-evaluation for their ability to induce cellular responses. Out of these, only F2 sub fraction belonging to the MW of 97.4-68 kDa stimulated remarkable lymphoproliferative and IFN-gamma responses in cured VL patients and in endemic controls. Similarly, significant lymphoproliferative responses and nitric oxide production were also noticed in cured Leishmania infected animals indicating an element of uniformity in responses between hamster and human. F2 sub fraction, when evaluated for its prophylactic efficacy with BCG against L. donovani challenge in hamster exhibited significant parasite inhibition in spleen (71.1%; p<0.001) and liver (68.2%; p<0.001) as compared to their unvaccinated counterpart. The vaccinated animals showed significant lymphoproliferative response and nitric oxide production but leishmania specific IgG level were suppressed. The results indicate the presence of immunostimulatory and protective molecules in F2 sub fraction which may further be exploited for the development of a vaccine against VL, hitherto an unrealized goal.     

中药对围绝经期大鼠脑组织中NOS mRNA的调节作用

目的:探讨中药更年乐佳液对围绝经期大鼠脑组织中一氧化氮合酶(Nitric oxide synthase,NOS)mRNA表达的影响。方法:将50只12～15月龄SD雌鼠随机分为5组:更年乐佳液高剂量组(乐高组)、更年乐佳液中剂量组(乐中组)、更年乐佳液低剂量组(乐低组)、空白对照组、尼尔雌醇组,每组10只。并以10只雌性成年未孕4～5月龄大鼠作为正常对照组。将麻醉好的动物取下丘脑组织,采用逆转录聚合酶链反应(RT-PCR)测定NOS mRNA的表达情况。结果:乐高组NOSmRNA表达总阳性率为80%;乐中组总阳性率为70%;尼尔雌醇组总阳性率为80%;正常对照组总阳性率为80%;均明显高于空白对照组50%(P<0.05)。而乐低组总阳性率仅为60%,与空白对照组相比无统计学差异(P>0.05)。结论:更年乐佳液可以促进神经元型NOS mRNA的表达。     

二甲苯对大鼠脑一氧化氮水平及一氧化氮合酶的影响

目的：探讨二甲苯对大鼠脑皮层匀浆上清液一氧化氮（ＮＯ）含量及海马区一氧化氮合酶（ＮＯＳ）活性的影响。方法：采用试剂盒法和ＮＡＤＰＨ组织学方法测定了ＮＯ含量和ＮＯＳ活性。结果：二甲苯接触组大鼠ＮＯ含量明显高于对照组（Ｐ＜０．０５），海马区ＮＯＳ阳性细胞数增加，但与对照组比较无显著性差异。结论：ＮＯ含量升高可能与二甲苯神经毒作用有关。海马区ＮＯＳ阳性细胞没有明显高于对照组而皮层匀浆上清液内ＮＯ含量升高的原因有待进一步阐明。     

葡萄皮提取物抗痴呆作用的实验研究

目的研究葡萄皮提取物抗痴呆效应及其机制。方法健康Wistar雄性大鼠,用D-半乳糖和亚硝酸钠腹腔注射建立拟痴呆模型,在造模同时分别给予生理盐水、脑复康、葡萄皮提取物低、中、高剂量干预。采用神经行为学及抗氧化类、胆碱能类和一氧化氮合酶、脑组织病理形态学和免疫组化等检测指标来进行观察、评价。结果葡萄皮提取物可明显改善拟痴呆模型大鼠的学习记忆功能,并可提高大鼠脑组织中超氧化物歧化酶、一氧化氮合酶、胆碱乙酰转移酶的活性,抑制丙二醛活性,减少模型组海马与皮质β-APP、β-AP阳性神经元细胞表达。结论葡萄皮提取物可抑制氧自由基形成,改善胆碱能神经系统的功能,增强大脑海马与皮层中一氧化氮合酶的活性,减少β-AP沉积斑块的形成,可能对早老性痴呆具有一定的防治作用。     

牛磺酸联合锌对急性缺氧小鼠大脑皮层NOS活力和nNOS蛋白表达的影响

目的观察牛磺酸和锌对急性缺氧小鼠大脑皮层一氧化氮合酶(NOS)和神经元型一氧化氮合酶(nNOS)阳性神经元的变化,探讨牛磺酸和锌抗脑缺氧的作用机制。方法复制小鼠急性缺氧模型,采用NADPHd组织化学和nNOS免疫组织化学方法,研究不同处理实验组急性缺氧小鼠皮层NADPHd和nNOS阳性神经元数量的变化。结果硫酸锌组小鼠标准缺氧耐受时间比生理盐水组延长33.06%,而牛磺酸+锌组小鼠标准缺氧耐受时间比硫酸锌组延长26.83%(P<0.05);硫酸锌组小鼠皮层NADPHd和nNOS阳性神经元的数量比生理盐水组明显减少,而牛磺酸+锌组小鼠皮层NADPHd和nNOS阳性神经元的数量比硫酸锌组明显减少(P<0.05)。结论急性缺氧时牛磺酸联合锌能够明显延长耐缺氧时间,其机制是通过降低皮层NOS活性和减少nNOS蛋白表达而发挥其抗脑缺氧作用。     

EFFECT OF ACUTE AND CHRONIC ETHANOL EXPOSURE ON THE RAT BRAIN OPIATE RECEPTOR FUNCTION

A reduction of the number and a decrease in the dissociationconstant of low-affinity opiate receptor binding sites for metenkephalinin brain membranes was noted in rats after chronic ethanol treatment.These changes were reversed with time, and at 48 hr after withdrawal,tissue from ethanol and pair-fed groups demonstrated similarbinding characteristics. Preincubation of the control rat brainmembrane fraction with the ultrafiltrate of the supernatantobtained from the brain membrane fraction of rats chronicallytreated with ethanol resulted in changes of affinity and numberof opiate receptor binding sites which resembled the changesobserved in rats after long-term treatment with ethanol. Acuteethanol administration (4 g/kg) was shown to eliminate the biphasiccharacter of Scatchard plots usually seen in control animals.Addition of ethanol to control brain membranes was also foundto after significantly the binding of ‘opiate’ peptidesto such membrane fractions.     

钙硒联合对铅暴露小鼠学习记忆能力影响

目的 探讨钙硒2种营养素联合应用对铅暴露小鼠学习记忆能力的影响.方法 3周龄清洁级昆明小鼠36只,随机分为3组:对照组,铅染毒组.钙硒联合干预组;在实验第20 d进行水迷宫实验,取小鼠的血、脑测定铅含量及NO含量、一氧化氮合酶(NOS)和诱导型一氧化氮合酶(iNOS)活性.结果 小鼠钙硒联合干预组血铅(469.59±58.31)μg/L和脑铅(0.460±0.034)μg/g含量高于对照组,低于铅染毒(885.02±105.77)μg/L和(1.290±0.270)μg/g组,差异有统计学意义(P<0.01);钙硒联合干预组脑NO含量为(8.29±1.22)μmol/g、NOS和iNOS活性分别为(4.39±0.98),(1.65±0.28),均低于对照组(10.27±2.27)ixmoVg,(6.22±1.25),(1.91±0.44)高于铅染毒组(5.66±1.04)μmol/g,(2.66±0.59),(0.67±0.14)差异有统计学意义(P<0.01);对照组、钙硒联合干预组小鼠水迷宫逃避潜伏期分别为(43.8±8.9),(44.1±11.7)s,短于铅染毒组的(85.9±23.5)s,差异有统计学意义(P<0.01),钙硒联合干预组与对照组差异无统计学意义.结论 钙和硒联合应用能有效地促进铅模型小鼠拮抗体内铅的毒性作用.     

Expression, but not activity, of neuronal nitric oxide synthase is regionally increased in the alcoholic brain

The regional distribution of nitric oxide synthase (NOS) was investigated in alcoholic post-mortem brains compared with brains of non-alcoholic control individuals. Total enzyme activity in 28 brain regions was determined using the [(3)H]l-citrulline formation assay, whereas Western blot analyses were used for semi-quantitative measurement of the neuronal isoform of NOS (nNOS). In the alcoholic brain, nNOS protein expression was increased in the following regions: frontal cortex (85%), the cingulate gyrus (294%), the nucleus accumbens (54%), the entorhinal cortex (85%) and the thalamus (51%). These increases were, however, not associated with higher total NOS activity. Interestingly, nNOS protein content was increased in the frontal cortex and the nucleus accumbens, brain regions which are suggested to be involved in the dopaminergic mesolimbic reward system. It is concluded that upregulation of signal transduction pathways, such as the adenosine 3',5'-monophosphate and the protein kinase C-dependent pathway, due to stimulation of G-protein-coupled neurotransmitter receptor regulation, as a form of functional tolerance, could be responsible for increased nNOS protein expression, and downregulation of NOS enzyme activity in these brain regions.     

INACTIVATION OF CEREBELLAR NITRIC OXIDE SYNTHASE BY ETHANOL IN VITRO

The N-methyl-D-aspartate receptor/nitric oxide synthase (NOS)/guanylatecyclase pathway, which plays a crucial role in synaptic plasticityin the brain, is modulated by ethanol. We studied the effectof ethanol in vitro on NOS in rat cerebellum and showed thatethanol (25–200 mM) inactivated NOS in a dose-dependentmanner. This inactivation was prevented by the biopterin cofactortetrahydrobiopterin (BH⁴) as well as by L-arginine, a NOS substrate,but not by NADPH. These results suggest that ethanol reducesNOS activity by modulating the conformation of the enzyme andthereby its stability, probably by interacting with the bindingsites of BH⁴ and/or of L-arginine. Our data also suggest thatinactivation of NOS may contribute to the decrease in the cGMPlevel, and thus may play a role in the pharmacological actionsof ethanol in vivo.     

Measurement of neurotoxic esterase activity in various subcellular fractions of hen brain and sciatic nerve homogenates and the effect of diisopropyl fluorophosphate (DFP) administration

Both the membrane and soluble compartments of brain and nerve tissue contain esterase activity. Experiments were done to assess neurotoxic esterase (NTE) activity in different subcellular fractions, obtained from brain and sciatic nerve homogenates. The inhibitory effect of diisopropyl fluorophosphate (DFP) on the various subcellular fractions was also studied. High levels of neurotoxic esterase activity was associated with the microsomal fractions (100,000g pellet) from brain and sciatic nerve extracts. All other subcellular fractions from brain homogenates yielded lower NTE activity. The comparative study of brain versus sciatic NTE activity demonstrated that substrate hydrolysis by sciatic microsomal fractions was much less than obtained with brain microsomes. The results showed that the percentage NTE of the total paraoxon-resistant activity was lower in sciatic nerve microsomal preparations than in brain microsomal fractions. Brain cytosol fractions (100,000g supernate) contained lower NTE activity than the corresponding sciatic nerve cytosol fraction. Myelin fractions were devoid of neurotoxic esterase activity. DFP had an overall inhibitory effect on microsomal and cytosol NTE activity. Based on the association of NTE with the microsomal fraction, neurotoxic esterase can be classified as a β-esterase. The results also indicate that quantitative differences exist both in the activity and in the quantity of NTE of the total paraoxon-resistant esterases of CNS and PNS.     

Ethanol withdrawal activates nitric oxide–producing neurons in anxiety-related brain areas

The present study investigated whether nitric oxide (NO)–producing neurons localized in brain areas related to anxiety are also activated after ethanol withdrawal. Male Wistar rats were subjected to an oral ethanol self-administration procedure, in which they were offered 6–8% (vol/vol) ethanol solution for a period of 21 days followed by abrupt discontinuation of the treatment. Control animals received control dietary fluid for similar periods of time. Twenty-four or 48 h after ethanol discontinuation, the animals were exposed to the open field for 10 min. Two hours later, their brains were removed and processed for Fos immunohistochemistry and nicotinamide adenine dinucleotide phosphate-diaphorase histochemistry (which is used to detect NO-producing neurons). Decreased exploratory activity was observed in animals subjected to 24-h withdrawal, characterized by a shorter distance traveled in the open field. Additionally, increased Fos expression was detected in brain areas, such as the cingulate and piriform cortices, several hypothalamic nuclei, amygdaloid nuclei, most subdivisions of the periaqueductal gray matter, and dorsal raphe nucleus (DRN). Ethanol withdrawal activated NO-producing neurons in the paraventricular nucleus (PVN) of the hypothalamus, dorsolateral periaqueductal gray matter (DLPAG), and DRN. The results show that ethanol withdrawal activates NO-producing neurons in the PVN, DLPAG, and DRN, which are brain areas implicated in the modulation of emotional, autonomic, and motor expression of anxiety-like behaviors.     

Prenatal alcohol exposure decreases the number of nitric oxide synthase positive neurons in rat superior colliculus and periaqueductal gray.

Because nitric oxide (NO) is involved in the development and refinement of axonal projections and synapses, it is of interest to know if developmental alcohol exposures affect NO producing neurons. Pregnant rats were fed artificial liquid diet throughout gestation as the only fluid or caloric source. The diet for experimental dams contained ethanol (6.7% v/v) while the pair-fed diet for control dams contained isocaloric maltose-dextrin instead of ethanol. This ethanol diet regime is known to produce peak blood alcohol concentrations of approximately 140 mg%. Cells stained histochemically for nitric oxide synthase (NOS) were counted at postnatal day 15 (P15) and 35 (P35) in cross-sections of the stratum griseum superficiale (SGS) of the superior colliculus (SC) and in the dorsolateral column of the periaqueductal gray (dlPAG). Compared to control tissues, alcohol caused the following effects: In the SC, the areal density of NOS+ neurons was decreased 24% at P15 but a similar decrease in means at P35 was not statistically significant (P=0.10); soma size was unaffected at either P15 or P35. In the dlPAG, both the areal density and the total number of NOS+ neurons per section were unaffected at P15 but were decreased at P35 (33% and 37% decreases); soma size was unaffected at either P15 or P35. The decrease in NOS+ neurons in the SC at P15 could be expected to have a negative impact on the refinement of neuronal connections while the decreases in NOS+ neurons in the dlPAG at P35 likely represent more permanent effects that could alter the function of that nucleus.     

不对称性二甲基精氨酸与早发型重度子痫前期关系的研究进展

不对称性二甲基精氨酸(ADMA)是内源性一氧化氮合酶(NOS)抑制剂,可抑制NOS活性,减少一氧化氮(NO)合成,导致血管内皮细胞功能障碍。早发型重度子痫前期(EOSP)存在胎盘形态学异常,而晚发型重度子痫前期(LOSP)则无明显胎盘形态学异常。因此,胎盘发生和形成异常,可能是导致EOSP发生的原因。该病患者胎盘血管内皮细胞的二甲基精氨酸二甲胺水解酶(DDAH2)表达水平下降,使ADMA降解减少;而血浆及胎盘ADMA水平增加,可竞争性抑制NOS活性,导致NO生成减少及其生物利用度降低,发生胎盘病变及胎盘血管内皮细胞功能障碍。由此可见,ADMA与EOSP胎盘病理改变的发生、发展密切相关,并已成为目前研究热点。笔者拟就ADMA在EOSP中的作用及其可能机制进行综述。     

Acute alcohol intake impairs lung inflammation by changing pro- and anti-inflammatory mediator balance.

Previous studies have shown that alcohol (ethanol [EtOH]) intoxication impairs lung immunity by affecting cytokines pivotal to the inflammatory process. The objective of this study was to test the hypothesis that acute alcohol intoxication impairs lung innate immunity by downregulating the expression of proinflammatory mediators while simultaneously upregulating anti-inflammatory mediators. EtOH was administered to the mice 0.5h prior to an intratracheal injection of Escherichia coli lipopolysaccharide (LPS). The animals were killed either 4 or 24h after LPS to recover plasma, lungs, and bronchoalveolar lavage fluid. Lung inflammatory cytokines tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1beta), IL-6, macrophage inhibitory factor (MIF), IL-10, TGF-beta, and receptors for TNF-alpha, IL-1beta, IL-6, and TGF-beta as well as glycoprotein (gp)130 and corticosterone (CS) levels were evaluated at mRNA and protein level. While the mRNA expression and the soluble TNF-Rp55 levels were significantly upregulated by EtOH, LPS-induced TNF-alpha activity, TNF-Rp55 mRNA expression, and soluble TNF-Rp55 levels were significantly suppressed. The LPS-induced expression of IL-1beta, IL-6, MIF, gp130, and receptors IL-1RI, IL-1RII, and IL-6Ralpha were also significantly impaired by EtOH. EtOH increased significantly the basal IL-10 activity at 3h, which continued to remain elevated even at 24h. The EtOH effect on IL-10 activity persisted even in LPS-challenged mice. EtOH and LPS augmented lung CS levels independently of each other. EtOH suppressed upregulation of TGF-beta1 mRNA expression by LPS and blocked completely LPS-induced TGF-beta1 secretion. In conclusion, the data suggest that the suppression of acute lung inflammation by EtOH intoxication is largely due to impairment by EtOH of proinflammatory cytokine signaling at the levels of cytokine expression and secretion as well as receptor expression and soluble receptor activity. The augmentation by EtOH of anti-inflammatory mediators' secretion most likely shifts the cytokine balance in the anti-inflammatory direction.     

Alcohol and Nitric Oxide Production by Cells of the Brain

The l-arginine–nitric oxide pathway is important to both physiological and pathologic brain events. Brain tissue contains cells able to express all known isoforms of nitric oxide synthase, the rate-limiting enzyme in nitric oxide (NO) production and release. Effects of ethanol on NO production may be important to ethanol modification of brain function. Recent studies support this idea and demonstrate diverse interactions. For example, acute ethanol treatment decreases NMDA- and cytokine-stimulated NO synthesis by cortical neurons and glia, respectively, but enhances cytokine-stimulated NO synthesis in blood–brain barrier endothelial cells and does not affect norepinephrine-stimulated NO synthesis in medial basal hypothalamus. Furthermore, chronic ethanol enhances NMDA-stimulated NO synthesis in cortical neurons, but more potently decreases cytokine-induced NO synthesis in glial cells. The mechanisms underlying these effects are partially understood and include changes in NOS-2 gene expression. These observations illustrate that ethanol selectively affects NO production by brain cells, which may relate to reported behavioral interactions, but the extend and direction of change depends on cell type and length of exposure.