alpha-Tocopherol protects against alpha-naphthylisothiocyanate-induced hepatotoxicity in rats less effectively than melatonin

The protective effect of alpha-tocopherol (alpha-Toc), which exerts antioxidant and anti-inflammatory actions, against alpha-naphthylisothiocyanate (ANIT)-induced hepatotoxicity in rats was compared with that of melatonin because orally administered melatonin is known to protect against ANIT-induced hepatotoxicity in rats through its antioxidant and anti-inflammatory actions. Rats intoxicated once with ANIT (75 mg/kg, intraperitoneal (i.p.)) showed liver cell damage and biliary cell damage with cholestasis at 24 h, but not 12 h, after intoxication. ANIT-intoxicated rats received alpha-Toc (100 or 250 mg/kg) or melatonin (100 mg/kg) orally at 12 h after intoxication. The alpha-Toc administration protected against liver cell damage in ANIT-intoxicated rats, while the melatonin administration protected against both liver cell damage and biliary cell damage with cholestasis. ANIT-intoxicated rats had increased hepatic lipid peroxide concentration and myeloperoxidase activity at 12 and 24 h after intoxication. ANIT-intoxicated rats also had increased serum alpha-Toc and non-esterified fatty acid (NEFA) concentrations at 12 and 24 h after intoxication and increased serum triglyceride and total cholesterol concentrations at 24h. The administration of alpha-Toc to ANIT-intoxicated rats increased the hepatic alpha-Toc concentration with further increase in the serum alpha-Toc concentration and attenuated the increased hepatic lipid peroxide concentration and myeloperoxidase activity and serum NEFA concentration at 24 h after intoxication. The melatonin administration did not affect the hepatic alpha-Toc concentration but attenuated the increased hepatic lipid peroxide concentration and myeloperoxidase activity and serum alpha-Toc, NEFA, triglyceride, and total cholesterol concentrations at 24 h after ANIT intoxication. These results indicate that orally administered alpha-Toc protects against ANIT-induced hepatotoxicity in rats possibly through its antioxidant and anti-inflammatory actions less effectively than orally administered melatonin.     

Antioxidant role of cellular reduced coenzyme Q homologs and alpha-tocopherol in free radical-induced injury of hepatocytes isolated from rats fed diets with different vitamin E contents.

Reduced coenzyme Q9 (CoQ9H2) and reduced coenzyme Q10 (CoQ10H2) as well as alpha-tocopherol (alpha-Toc) are known to be potent lipid-soluble antioxidants in mammalian tissues. Reduced coenzyme Q homolog (CoQnH2) appears to show antioxidant activity independent of that of alpha-Toc (Matsura, T., Yamada, K. and Kawasaki, T. (1992) Biochim. Biophys. Acta 1123, 309-315). To further confirm this, we have studied the antioxidant role of cellular CoQnH2 and alpha-Toc using hepatocytes isolated from rats fed diets containing deficient, sufficient, and excess amounts of vitamin E (VE). Cellular damage was induced with a hydrophilic radical initiator, 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH). The concentration of alpha-Toc in VE-deficient hepatocytes was approximately 1/12 that in VE-sufficient hepatocytes, whereas the concentration of alpha-Toc in VE-excess hepatocytes was approximately 7-fold that in VE-sufficient hepatocytes. The molar ratios of alpha-Toc to CoQnH2 (CoQ9H2 plus CoQ10H2) in VE-deficient, sufficient and excess cells were 0.03, 0.33 and 2, respectively. In the hepatocytes in these three dietary groups, alpha-Toc status had little effect on the concentration of CoQ homologs. These hepatocytes were incubated with 50 mM AAPH for 4 h. The cell viability in all groups of hepatocytes decreased rapidly after 3 h of AAPH treatment, and was associated with the increase of lipid peroxides. The loss of cell viability and the increase of lipid peroxidation in VE-deficient cells were more pronounced than those in the hepatocytes of the other two groups. The endogenous CoQ9H2 content of each group of hepatocytes decreased linearly with a reciprocal increase in oxidized CoQ9 after addition of AAPH, whereas the decrease of endogenous CoQ10H2 in each group during AAPH treatment was much less than that of endogenous CoQ9H2. alpha-Toc in the three VE dietary groups of hepatocytes was also consumed without a time lag after addition of AAPH, and it was not spared by CoQnH2, even in VE-deficient cells where the CoQnH2 concentration was 38-fold that of alpha-Toc. These results indicate that CoQnH2, especially. CoQ9H2, is a lipid-soluble antioxidant, which is as effective as alpha-Toc in rat hepatocytes under the conditions employed in this study, and acts independently of alpha-Toc to inhibit lipid peroxidation.     

Dietary docosahexaenoic acid enhances ferric nitrilotriacetate-induced oxidative damage in mice but not when additional alpha-tocopherol is supplemented

Weaning mice were fed a diet supplemented with beef tallow (BT) or BT plus docosahexaenoic acid (DHA) containing 100 mg alpha-tocopherol/kg (alpha-Toc100) or 500 mg alpha-tocopherol/kg (alpha-Toc500) for 4 wk to modify membrane fatty acid unsaturation, and then were administered ferric nitrilotriacetate (Fe-NTA). The mortality caused by Fe-NTA was higher in the group fed the DHA (alpha-Toc100) diet than in the BT diet groups but the DHA (alpha-Toc500) diet suppressed this increase. Serum and kidney alpha-tocopherol contents were slightly influenced by the dietary fatty acids but not significantly. These results indicate that the increased unsaturation of tissue lipids enhances oxidative damage induced by Fe-NTA in mice fed DHA (alpha-Toc100) but not when additional alpha-tocopherol is supplemented. The apparent discrepancy between the observed enhancement by dietary DHA of oxidative damage and the beneficial effects of dietary DHA on the so-called free radical diseases is discussed in terms of strong bolus oxidative stress and moderate chronic oxidative stress.     

Inhibitory effects of vitamin E on endothelial-dependent adhesive interactions with leukocytes induced by oxidized low density lipoprotein

Leukocyte-endothelial cell interactions, which are mediated by various adhesion molecules, are a crucial event in inflammatory reactions including atherosclerosis. Alpha-tocopherol (alpha-Toc) has been used for protection and therapy of vascular diseases because of its antioxidant activity. The objective of the present study was to determine effect of alpha-Toc on endothelial-dependent adhesive interactions with leukocytes elicited by oxidized low density lipoprotein (oxLDL). Incubation of HUVEC with oxLDL (100 microg/mL) increased expression of proteins and messenger RNA of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) on enzyme immunoassay and northern blotting assay; pretreatment with alpha-Toc reduced in a dose dependent manner. Adherence of polymorphonuclear leukocytes (PMN) or mononuclear leukocytes (MNC) to oxLDL-activated HUVEC was much increased compared with that to unstimulated HUVEC. Treatment of HUVEC with alpha-Toc, monoclonal antibody to ICAM-1 or VCAM-1 inhibited adherence of PMN or MNC in a dose dependent manner. These results suggest that alpha-Toc works as anti-atherogenic agent through inhibiting endothelial-dependent adhesive interactions with leukocytes induced by oxLDL.     

Anisotropy measurements of intrinsic fluorescence of prenyllipids reveal much higher mobility of plastoquinol than alpha-tocopherol in model membranes

As an alternative to a fluorescent probe approach, the intrinsic fluorescence of reduced forms of prenylquinones has been exploited, which offers a convenient means of determining directly motional properties of these molecules. The steady-state fluorescence anisotropy measurements of plastoquinols (PQH(2)) and alpha-tocopherol (alpha-Toc) incorporated into phospholipid liposomes have been performed. The effect of prenyllipid concentration, PQH(2) side chain length and the composition of the membranes has been studied. For the data interpretation, the fundamental anisotropy of alpha-Toc, PQH(2), ubiquinol-10 and alpha-tocopherolquinol, as well as the angles between the absorption and emission transition moments have been also determined. It was concluded that alpha-Toc shows very low mobility in the lipid bilayer, whereas PQH(2)-9 displays significant motional freedom in dipalmitoylphosphatidylcholine vesicles and even higher in egg yolk lecithin membranes.     

Small molecular antioxidants effectively protect from PUVA-induced oxidative stress responses underlying fibroblast senescence and photoaging

Exposure of human fibroblasts to 8-methoxypsoralen plus ultraviolet-A irradiation (PUVA) results in stress-induced cellular senescence in fibroblasts. We here studied the role of the antioxidant defense system in the accumulation of reactive oxygen species (ROS) and the effect of the antioxidants alpha-tocopherol, N-acetylcysteine, and alpha-lipoic acid on PUVA-induced cellular senescence. PUVA treatment induced an immediate and increasing generation of intracellular ROS. Supplementation of PUVA-treated fibroblasts with alpha-tocopherol (alpha-Toc), N-acetylcysteine (NAC), or alpha-lipoic acid (alpha-LA) abrogated the increased ROS generation and rescued fibroblasts from the ROS-dependent changes into the cellular senescence phenotype, such as cytoplasmic enlargement, enhanced expression of senescence-associated-beta-galactosidase and matrix-metalloproteinase-1, hallmarks of photoaging and intrinsic aging. PUVA treatment disrupted the integrity of cellular membranes and impaired homeostasis and function of the cellular antioxidant system with a significant decrease in glutathione and hydrogen peroxide-detoxifying enzymes activities. Supplementation with NAC, alpha-LA, and alpha-Toc counteracted these changes. Our data provide causal evidence that (i) oxidative stress due to an imbalance in the overall cellular antioxidant capacity contributes to the induction and maintenance of the PUVA-induced fibroblast senescence and that (ii) low molecular antioxidants protect effectively against these deleterious alterations.     

Deletion of vitamin E enhances phenotype of Alzheimer disease model mouse

Increased oxidative damage is a prominent and early feature in Alzheimer disease (AD). However, whether it is a primary cause or merely a downstream consequence in AD pathology is still unknown. We previously generated alpha-tocopherol transfer protein knockout (Ttpa-/-) mice, in which lipid peroxidation in the brain was significantly increased by complete depletion of alpha-tocopherol (alpha-Toc). Here we crossed AD transgenic (APPsw) model mice (Tg2576) with Ttpa-/- mice. The resulting double-mutant (Ttpa-/- APPsw) mice showed earlier and more severe cognitive dysfunction in the Morris water maze, novel-object recognition, and contextual fear conditioning tests. They also showed increased amyloid beta-peptide (Abeta) deposits in the brain by immunohistochemical analysis, which was ameliorated with alpha-Toc supplementation. In this report we provide clear evidence indicating that chronic lipid peroxidation due to alpha-Toc depletion enhances AD phenotype in a mouse model.     

Effect of alpha-tocopherol on the oxidative modification of apolipoprotein E in human very-low-density lipoprotein

The oxidative modification of apolipoprotein (apo) E and lipid peroxidation in human very-low-density lipoprotein (VLDL) induced by peroxynitrite and cupric ions in vitro were strongly suppressed by enrichment with alpha-tocopherol (alpha-Toc; 170 microM). Alpha-Toc also suppressed the decrease in the heparin-binding activity of apoE in the VLDL oxidation. These results suggest that alpha-Toc protected apoE in VLDL from oxidative stress.     

High-cholesterol diets induce changes in lipid composition of rat erythrocyte membrane including decrease in cholesterol,increase in alpha-tocopherol and changes in fatty acids of phospholipids

Effects of high dietary cholesterol on erythrocyte membrane lipids were studied. Feeding rats with a diet containing 0.5% cholesterol and 0.15% sodium cholate for two weeks induced changes in erythrocyte membrane lipids including a decrease in cholesterol, an increase in alpha-tocopherol (alpha-Toc) and changes in the fatty acid composition of phospholipids. Oleic acid and linoleic acid increased, while arachidonic acid decreased in phosphatidylcholine. Saturated fatty acids decreased and unsaturated fatty acids increased in phosphatidylethanolamine. Almost the same changes in membrane lipids were also noted after six weeks of feeding rats with the diet. A diet containing 0.5% cholesterol but without sodium cholate caused a decrease in erythrocyte cholesterol and an increase in erythrocyte alpha-Toc after two weeks of feeding, as compared to the basal diet, indicating that high dietary cholesterol, but not sodium cholate, was responsible for these changes in the erythrocyte membrane.     

Characterization of monochloramine toxicity on PC12 cells and protective effect of tocopherol via antioxidative function

Monochloramine (NH(2)Cl) is a physiological oxidant produced by activated neutrophils. In the present work, we studied the underlying mechanism of cytotoxic effects of NH(2)Cl on an undifferentiated rat pheochromocytoma PC12 cell line and the protective effects of antioxidants. The cells treated with 100 microM NH(2)Cl exhibited signs of apoptotic cell death such as phosphatidylserine exposure and caspase activation. To understand the mechanism of NH(2)Cl cytotoxicity, we examined the effect of various kinds of antioxidants including alpha-tocopherol (alpha-Toc) and beta-tocopherol (beta-Toc). These antioxidants exerted a protective effect against NH(2)Cl-induced cell death, and alpha-Toc exhibited the most potent inhibitory effect among the antioxidants used. A loss of cellular glutathione was observed in the cells treated with 100 microM NH(2)Cl. The formation of reactive oxygen species (ROS) was also measured using the fluorescent probe dichlorofluorescin diacetate. The fluorescence intensity increased prior to cell death and an antioxidant, such as alpha-Toc, suppressed the increase in ROS. Interestingly, beta-Toc also exerted similar inhibitory effects on cytotoxicity and caspase activation. These results suggest that free radical mediated process is involved in NH(2)Cl-induced PC12 cell death and that tocopherols inhibit this cell death via antioxidative function.     

Studies of the metabolism of alpha-tocopherol stereoisomers in rats using [5-methyl-(14)C]SRR- and RRR-alpha-tocopherol

We investigated the distribution and metabolism of SRR-alpha-tocopherol (SRR-alpha-Toc), synthetic alpha-Toc compared with RRR-alpha-Toc, in rats after a single oral administration of 2 mg (20 microCi) SRR- and RRR-alpha-[5-methyl-(14)C]Toc. In the liver, there was no difference in the recovery of radioactivity until 12 h after administration, and it reached a maximum of 4.4% of the dose after 12 h, but in other tissues, radioactivity derived from RRR-alpha-Toc was clearly higher than that derived from SRR-alpha-Toc after 12 h. For 96 h after administration, urinary excretions of SRR-alpha-Toc were 7.8% of the dose and significantly greater than that of RRR-alpha-Toc, which was 1.3% of the dose. On the other hand, total fecal excretions of SRR- and RRR-alpha-Toc were 87.6% and 83.0%, respectively. Therefore, radioactivity in the urine was assumed to have transferred out of the liver. Furthermore, the urine samples were hydrolyzed with 3 N methanolic HCl and analyzed by high performance liquid chromatography (HPLC) and liquid chromatography/mass spectrometry. The results showed that about 73% of the total radioactivity injected into HPLC was found to be 2,5,7, 8-tetramethyl-2-(2'-carboxyethyl)-6-hydroxy chroman (alpha-CEHC), as well as RRR-alpha-Toc. Thus, there is no difference between SRR-alpha-Toc and RRR-alpha-Toc in metabolic pathways, and it is suggested that SRR-alpha-Toc discriminated in the liver is rapidly metabolized by the liver and excreted as the conjugate of alpha-CEHC in the urine.     

Regulation of rat liver phosphoenolpyruvate carboxykinase (GTP) messenger ribonucleic acid activity by N6, O2'-dibutyryladenosine 3',5'-phosphate

N6,O2'-Dibutyryladenosine 3',5'-phosphate (Bt2cAMP) induces the synthesis of the gluconeogenic enzyme, phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.32), in rat liver by increasing the activity of messenger ribonucleic acid (mRNA) coding for this enzyme (mRNAPEPCK) more than 20-fold (from less than 0.01% to greater than 0.20% of total mRNA activity) as determined by using in vitro translation systems which measure only active mRNAPEPCK. The increase in mRNAPEPCK activity could result from increased synthesis, increased processing, or decreased inactivation rates. Actinomycin D and cordycepin inhibit mRNAPEPCK induction by 89% and 70%, respectively, a result that indicates a requirement for ongoing RNA synthesis but that does not distinguish which of these steps is regulated by cAMP. We have employed a kinetic approach, not involving RNA synthesis inhibitors, to determine the half-life of mRNAPEPCK both during a period of deinduction following glucose feeding and during a subsequent induction by Bt2cAMP. An estimated half-life of 20 +/-5 min during both of these periods indicates that Bt2cAMP has no effect on the rate of inactivation of mRNAPEPCK. We conclude that Bt2cAMP effects the increase in activity of mRNAPEPCK by promoting its synthesis or processing.     

Stopped-flow investigation of antioxidant activity of estrogens in solution

A kinetic study of the reaction between estrogens (female hormone) and substituted phenoxyl radical has been performed, as a model for the reactions of estrogens with lipid peroxyl radical in biological systems. The rates of reaction of estrogens (estrone 1, estradiol 2, 2-methoxyestrone 3, 3-methoxyestrone 4, and 2-hydroxyestrone 5) with substituted phenoxyl radical in benzene have been determined spectrophotometrically, using stopped-flow technique. The second-order rate constants, k2, obtained are 84 M-1.s-1 for 1, 138 M-1.s-1 for 2, 520 M-1.s-1 for 3, less than 10(-4) M-1.s-1 for 4, and 2.6 X 10(5) M-1.s-1 for 5 at 25.0 degrees C. 2-Hydroxyestrone 5 was found to be 2.9-times more active than alpha-tocopherol, which has the highest antioxidant activity among natural tocopherols. The order of magnitude of k2 value (1 less than 2 less than 3 less than alpha-Toc less than 5) is in agreement with that of in vitro tests of their antioxidant activities, as measured by the inhibition of lipid peroxidation. Further, similar measurements have been performed for the reaction between the above estrogens 1-5 and tocopheroxyl 6 in benzene solution. It was found that the estrogens having an OH group at the aromatic ring have an ability to regenerate the tocopheroxyl 6 to tocopherol. Especially, the 2-hydroxyestrone 5 showed about three orders of magnitude higher reactivity than ascorbic acid.     

Comparative Induction of Nitrate and Nitrite Uptake and Reduction Systems by Ambient Nitrate and Nitrite in Intact Roots of Barley (Hordeum vulgare L.) Seedlings

The induction by ambient NO3- and NO2- of the NO3- and NO2- uptake and reduction systems in roots of 8-d-old intact barley (Hordeum vulgare L.) seedlings was studied. Seedlings were induced with concentrations of NaNO3 or NaNO2 ranging from 0.25 to 1000 [mu]M. Uptake was determined by measuring the depletion of either NO3- or NO2- from uptake solutions. Enzyme activities were assayed in vitro using cell-free extracts. Uptake and reduction systems for both NO3- and NO2- were induced by either ion. The Km values for NO3- and NO2- uptake induced by NO2- were similar to those for uptake induced by NO3-. Induction of both the uptake and reduction systems was detected well before any NO3- or NO2- was found in the roots. At lower substrate concentrations of both NO3- and NO2- (5-10 [mu]M), the durations of the lag periods preceding induction were similar. Induction of uptake, as a function of concentration, proceeded linearly and similarly for both ions up to about 10 [mu]M. Then, while induction by NO3- continued to increase more slowly, induction by NO2- sharply decreased between 10 and 1000 [mu]M, apparently due to NO2- toxicity. In contrast, induction of NO3- reductase (NR) and NO2- reductase (NiR) by NO2- did not decrease above 10 [mu]M but rather continued to increase up to a substrate concentration of 1000 [mu]M. NO3- was a more effective inducer of NR than was NO2-; however, both ions equally induced NiR. Cycloheximide inhibited the induction of both uptake systems as well as NR and NiR activities whether induced by NO3- or NO2-. The results indicate that in situ NO3- and NO2- induce both uptake and reduction systems, and the accumulation of the substrates per se is not obligatory.     

Periodically discontinuous induction of bone marrow stem cells toward osteogenic differentiation in vitro

This paper reveals that a discontinuous in vitro induction, namely, the periodic presence and absence of foreign induction factors, might be, under a certain condition, more effective to stimulate stem cells' differentiation than a continuous induction. Bone marrow stem cells (BMSCs) derived from Sprague Dawley rats were employed to examine the effects of discontinuous additions of osteogenic supplements with a series of alternate frequency in contrast to those with continuous induction or no induction. The results demonstrated that a suitable discontinuous induction was more able to achieve osteogenesis than not only no induction but also the associated continuous induction. Additionally, the osteogenic supplements were confirmed to enhance cell differentiation but suppress cell proliferation. So, the combination of differentiation extent per cell and cell number accounts for the "unexpected" good osteogenic effect of the discontinuous induction. The induction effect was found to be dependent upon alternate frequency, and the optimum alternate period in our experimental systems was determined to be around 4 days. Since it is very common to change culture medium every 2-4 days, such a strategy of discontinuous induction does not bring any extra manual work but reduces the consumption of foreign induction factors and significantly enhances the global differentiation efficacy. Our work thus affords a convenient and practical approach to achieve differentiation of BMSCs, which might be useful for potential large-scale culture and differentiation of stem cells. Meanwhile, the existence of optimum frequency implies some unknown inherent rhythms of cell proliferation and differentiation.     

Evidence for Substrate Induction of a Nitrate Efflux System in Barley Roots

Induction of an NO3- efflux system in intact barley (Hordeum vulgare L.) roots was demonstrated. Since the measurement of NO3- efflux is dependent on its accumulation, experiments were devised to facilitate accumulation under noninducing conditions. This was accomplished by incubating seedlings in 10 mM NO3- in the presence of RNA and protein synthesis inhibitors. Under these conditions NO3- uptake is mediated by constitutive high- and low-affinity transport systems. Control roots were incubated with 1.0 mM NO3-. This resulted in the accumulation of similar levels of NO3- in both treated and control roots; however, cytoplasmic NO3- efflux from inhibitor-treated roots was much lower than from control roots. Following a brief lag period, efflux rates increased rapidly in the presence of NO3- for 8 to 12 h. The NO3- efflux system was also induced by ambient NO2-. After induction the efflux system was relatively stable in the presence of RNA and protein synthesis inhibitors as long as NO3- or NO2- was present. These results suggest that NO3- efflux may be an inducible system requiring both RNA and protein synthesis, as does induction of the uptake system. The efflux system, however, has a much slower turnover rate than the uptake system.     

Four distinct photoreceptors contribute to light-induced side branch formation in the moss <Emphasis Type="Italic">Physcomitrella patens</Emphasis>

Side branch formation in the moss, Physcomitrella patens, has been shown to be light dependent with cryptochrome 1a and 1b (Ppcry1a and Ppcry1b), being the blue light receptors for this response (Imaizumi et al. in Plant Cell 14:373, 2002). In this study, detailed photobiological analyses were performed, which revealed that this response involves multiple photoreceptors including cryptochromes. For light induction of branches, blue light of a fluence rate higher than 6 μmol m⁻² s⁻¹ for period longer than 3 h is required. The number of branches increased with the increase in fluence rate and in the irradiation period. The number of branches also increased when red light was applied together with the blue light, although red light alone had a very few effect. By partially irradiating a cell, both receptive sites for blue and red light were found to be located around the nucleus. Further, both red and blue light determine the positions of branches being dependent upon the vibration plane of polarized light. Red light control of branch position was nullified by simultaneous far-red light irradiation. A blue light effect on branch position was not found in lines with disrupted phototropin genes. Thus, dichroic phytochrome and phototropin, possibly on the plasma membrane, regulate branch position. These results indicate that at least four distinct photoreceptor systems, namely, cryptochromes and red light receptor around or in the nucleus, dichroic phytochrome and phototropin around the cell periphery, are involved in the light induction of side branches in the moss Physcomitrella patens.     

A comparison of chromosome aberration induction by 25 compounds tested by two Chinese hamster cell (CHL and CHO) systems in culture

Twenty-five chemicals were tested for the induction of chromosomal aberrations in 2 cultured mammalian cell systems, Chinese hamster lung cells (CHL) and Chinese hamster ovary cells (CHO). This study was carried out to provide a data set that would permit an assessment of the extent to which the 2 systems agree in the results produced. Results presented for the 2 systems in this paper are not based on the same criteria but rather on the criteria standardly used in each of the systems. In tests conducted in the absence of S9 mix, 7 chemicals gave positive results in both systems and 12 were negative in both. In tests with S9 mix, 5 were positive in both systems and 9 were negative in both. When the overall results including tests both with and without S9 mix were considered, the 2 systems agreed on 15 results, 11 positives and 4 negatives. A review of the test conditions and data suggests that disagreements in test results were more often due to differences in the protocols used in these 2 systems than to a difference in the sensitivities of the 2 cell lines.