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Induction of Cytochrome P-45OIA1 Activity in Response to Sublethal Stresses in Microcarrier-Attached Hep G2 Cells Naheed A. Mufti?and Michael L. Shuler* School of Chemical Engineering, 340 Olin Hall, Cornel1 University, Ithaca, New York 14853
Cell damage for cells grown on microcarriers in suspension is a critical problem for scale-up of microcarrier reactors. In order to study cell damage as a mechanistic process, a cellular response that is more sensitive than changes in growth and death rates and would be more closely related to cell regulatory mechanisms would be advantageous. We have observed the induction of a specific cytochrome P-450 monooxygenase, P-45OIA1 (CYPlAl), to be a sensitive method for assessing the response of microcarrier-attached Hep G2 cells to stress resulting from hydrodynamic shear and oxygen deprivation. The kinetics of induction and amount of CYPlAl formed in response to subtle shear stress, moderate shear, and hypoxia are described. Increased stress results in increased CYPlAl formation.
Introduction The design of bioreactors for anchorage-dependent cells can be facilitated by using microcarrier cultures. The advantages of agitated microcarrier cultures include more uniform environments than other culture systems for attached cells, increased mass transfer of oxygen and other nutrients, and simpler process monitoring (Papoutsakis, 1991). However, both agitation and aeration can result in cell damage. The mechanisms of cell damage in agitated microcarrier cultures include interactions of the cells on the microcarriers with other beads, with the surrounding fluid, or with the vessel and impeller blades (Croughan et al., 1987; Cherry and Papoutsakis, 1988; Papoutsakis, 1991). Cell damage in microcarrier cultures has typically been evaluated by comparing growth and death rates of cells at varying agitation intensities (Croughanet al., 1988; Lakhotia and Papoutsakis, 1992). However, much lower levels of shear may alter cellular regulation complicating scale-up of such cultures. Low levels of shear as experienced under physiological conditions can significantly alter production of cellular metabolites and proteins (Nollert et al., 1989, 1991, 1992). A better understanding of induction of changes in cell regulation by sublethal levels of stress is needed. Microcarrier cultures can also be used in the design of in vitro toxicity studies due to the ease of obtaining kinetic data for dose-response curves and for the advantages listed previously. In the process of designing a bioreactor to evaluate the effects of 2,3,7,84etrachlorodibenzo-p-dioxin (TCDD)in a human hepatoma cell line, Hep G2, we observed the increase of cytochrome P-45OIA1 (CYPlAl) activity in response to physical stresses on the cells. CYPlAl induction is a marker response for TCDD exposure (Lipp et al., 19921, but in the absence of TCDD, there was significant CYPlAl induction in the microcarrier-attached cells. We have presented evidence that the Ah receptor (AhR)is involved in the response to shear (Mufti et al., 1995);the binding of TCDD to a Ah receptor complex is known to be part of the mechanism for induction of CYPlAl (Safe, 1986; Probst et al., 1993). Although there is yet no direct evidence that shear acts at the level of gene induction, it is plausible that shear
* To whom correspondence should be addressed. Current address: SRI International, Physical Sciences Div., Menlo Park, CA 94025. 8756-7938/95/3011-0659$09.00/0
may act at the level of gene induction since the Ah receptor is involved in both the shear and TCDD responses. CYPlAl is a phase I drug-metabolizing enzyme that has been shown to be induced when cells are exposed to environmental contaminants such as dioxins and PCB's. Furthermore, CYPlAl has been shown to be involved in the oxidation of arachidonic acid (Rifkind et al., 1990, 1994 Nakai et al., 1992) leading to possible metabolites that have been implicated in intracellular responses including cell proliferation (Sellmayer et al., 1991) and c-fos gene expression (Sellmayer et al., 1991; Rao et al., 1993). In this paper, we report the increase of CYPlAl activity in Hep G2 cells, attached to Cytodex 3 microcarriers, both in spinner flask and in a bioreactor designed for in vitro toxicity studies, as a response to sublethal levels of shear and to anoxia.
Materials and Methods Materials. Crystal violet, citric acid, and Cytodex 3 microcarriers were purchased from Sigma Chemical Co. (St. Louis, MO). MEM, S-MEM (suspension-minimum essential medium), trypsin, and FBS were purchased from Gibco BRL Life Technologies (Grand Island, NY). Ethoxyresorufin (EOR) and resorufin were purchased from Molecular Probes (Eugene, OR). Spinner flasks (50 mL) and spinner plates were purchased from Bellco (Vineland, NJ). Cell Culture. Hep G2 cells, obtained from ATCC (Rockville, MD), is a continuous human hepatoma cell line. The Hep G2 cell line was derived from a tumor biopsy (Aden et al., 1979). The Hep G2 cells were grown in S-MEM supplemented with 10%FBS at 37 "C in 95% air and 5% COZin T-flask cultures. The pH was 7.5 0.1. Cells were passaged using split ratios of 1:4 at 6-day intervals. Cells were used for experiments within 20 passages to ensure cell line stability. One T-75 flask was used to inoculate one 50-mL spinner flask with a working volume of 40 mL of S-MEM and a microcarrier density of 3 g/L using the procedure of Forestell et al. (1992).The spinner flasks were equipped with a round-tipped impeller with 0.6 cm diameter and 4 cm length (see Figure 1A). The impeller was positioned in an impeller shaft with a height of 3.5 cm and a diameter of 1.3 cm. The bottom of the impeller shafi was 0.5 cm from the bottom
0 1995 American Chemical Society and American Institute of Chemical Engineers
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660 Cas Exchange
.
n
Silicone-Coated
!e! 0.5 cm
!-
1.3 cm
TCDD +
@
Other Tissue
Air + ,5%CO,
1
Liver Q-”artmer\f
Figure 1. (A) Schematic diagram of a 50-mL spinner flask. (B) Schematic diagram of the two-compartment reactor system developed to study the effects of TCDD in the human hepatoma cell line Hep G2.
of the flask, the center of the impeller was placed 1 cm from the bottom of the flask, and there was 0.5 cm between the tip of the impeller and the vessel wall. Shear effects would be expected to vary with changes in impeller size, shape, and placement. Porous foam closures were used to ensure adequate oxygen supply. Medium was exchanged in the spinner flasks after 2 days. Cells were grown in spinner flask at 65 rpm for 3 days before being used in an experiment at which point the cells on the microcarriers were approximately 80% confluent. Spinner Flask Experiments. Cells, together with the medium, were transferred from the growth spinner flask to a freshly washed and autoclaved flask (a “clean” flask) and placed in the same incubator and on the same spinner plate at 65 rpm for the standard control experiments. The spinner flask in which the cells were grown prior to transfer is referred to as a “conditioned flask”. Such flasks may have compounds from cell growth attached to the surfaces of the flask. The cells were transferred slowly down the arm of the spinner flask with a standard 10-mL pipet. Other methods of transferring that were tested included a wide-bore pipet and gently pouring down the arm of the receiving flask. The effects of increasing shear on the response were studied by either increasing the agitation rate or by using a modified spinner flask equipped with a flat edge baffle, of length 4.5 cm and width 1.5 cm, mounted on the impeller shaft. The effect of decreasing the oxygen supply to the cells was studied by using a spinner flask with caps instead of the foam closures and opening one cap by one-quarter turn. Reactor Experiments. Figure 1B illustrates the two-
compartment reactor designed for in vitro toxicity testing. This system was intended to mimic human physiology with a “Liver Compartment’’ containing cells and an “Other Tissues Compartment” for the well-perfused organs (see Mufti, 1995). The total working volume of the reactor is 300 mL while the volume of the cell compartment is 80 mL with a microcarrier density of 3 g/L as in the spinner flask experiments. The microcarrier-attached Hep G2 cells are contained within the first compartment by using a nylon mesh to separate the cells from the medium recirculation region located above the mesh. The remaining medium was in the “Other Tissue Compartment” and tubing which served the purpose of having a system with a volume equivalent to one-tenth of the human plasma volume for the dioxin in vitro toxicity studies. The separation of the Hep G2 cells in the “Liver Compartment” from the medium in the “Other Tissue Compartment’’was necessary to observe the distribution of dioxin in the system and the resultant CYPlAl induction. The flow rate chosen for recirculating the medium between the two compartments was 80 mL/ min. This flow rate is based on the physiological flow rate of blood flow from the heart to the liver and thus required that the medium exchange in the “Liver Compartment” occur above the mesh such that the cells would not travel through the entire system. The microcarriers were maintained in suspension by agitating at 65 rpm. Gas exchange occurred in the “Other Tissues Compartment” using 95% air/5% CO2 with the vessel being mixed at 150 rpm. Cells were transferred from the spinner flasks to the inoculation flask by pipetting followed by gravity flow into the reactor, and the supplemental medium was added to the second vessel. Microcarriers and cells could be sampled directly from the reactor. The two-compartment reactor was operated in two ways. In the first case, the cells were inoculated in the reactor for 12 h but the cell volume was controlled such that there was a liquid-air interface below the mesh. After 12 h, the remaining medium was added and the pumps were started. In the second case, the cells and total volume of medium were added and the pumps were initiated immediately. Determination of Cell Number. The method of van Wezel was used to determine cell number (van Wezel, 1973). Aliquots 0.5 mL of immobilized cells were washed twice with D-PBS (Dulbecco’sphosphate buffered saline). The D-PBS was then removed, and the cells were resuspended in 0.1 M citric acid. After 10 min, cells were stained with a 1:lratio of cell suspension to 0.15% crystal violet in 0.1 M citric acid. After staining for 1 h, cells were counted using a hemacytometer. Measurement of CYPlAl Activity. CYPlAl activity was measured as ethoxyresorufin-0-dealkylase(EROD) production rate by a modification of the method of Burke and Mayer for suspended cells (Burke and Mayer, 1974; Gentest, 1990). Aliquots 0.5 mL of cells were washed twice with D-PBS. The D-PBS was removed, and the cells were resuspended in 2 mL of reaction buffer (0.05 M Trizma base, 0.01 M MgC12, 0.2 M sucrose). EROD production rate was measured after addition of 2 pL of 0.5 mM EOR. The reaction was performed in a SLMAminco AB2 spectrofluorometer (SLM-Aminco,Urbana, IL) at 37 “C in a stirred cuvette holder. The change in fluorescence intensity with respect to time was converted to picomoles of resorufin formed per minute using a standard curve for resorufin fluorescence intensity.
Results The transient induction of CYPlAl activity in Hep G2 cells grown on microcarriers is shown in Figure 2. Microcarrier-attached cultures were grown for 3 days in
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661 Table 1. Comparison of Different Methods of Cell Transfer and Vessels for Cell Transfers on the Maximum Induction of CYPlAl in Hep G2 Cells Immobilized on Cytodex 3 Microcarriers" EROD formation percent of rate (pmol of standard resorufin formed/ control transfer method/transfer flask (min/106cells))b (S) A. Standard Controlc 1.60 h 0.33 transfer by 10-mL standard pipet 100 to clean spinner flask at 65 rpm B. Altered Method of Transfer to a Clean Flask pipet into bottom of flask 1.93 120 wide-bore 10-mL pipet 1.39 87 1.49 pour from top of the flask 93 pour from flask side arm 1.40 88 C. Altered Flasks cells grown in a siliconized flask 1.75 110 prior to transfer into a clean spinner flask at 65 rpm 1.77 square impeller (1 = 4.5 cm) in 110 clean recipient flask 19d conditioned recipient flask 0.3 19d conditioned siliconized 0.3 recipient flask 0.3 19d clean recipient spinner flask without stirring (V = 20 mL) Od 0 as recipient T-flask (10"-75)
1 E o 0
&
O
2
4
6
8
1 0 1 2 1 4
Time (h)
Figure 2. Kinetics of CYPlAl induction by transferring Hep G2 cells, attached to Cytodex 3 carriers, grown previously for 3 days in a 50-mL spinner flask and then transferred to a clean spinner flask. Each point represents the average of duplicate samples.
standard 50-mL spinner flasks prior to transfer with a standard 10-mL pipet to a freshly washed and autoclaved 50-mL spinner flask (or "clean" flask) with stirring at 65 rpm. This experiment is referred to as the "standard control". The process of transfer to a clean spinner flask with stirring at 65 rpm appears to act as a stress on the cells leading to increased CYPlAl activity (as detennined by EROD formation rate). There is a dramatic and very reproducible increase in CYPlAl activity in the Hep G2 cells with a peak occurring 8 h after the transfer to the "clean" spinner flask followed by a rapid decline in CYPlAl activity after 12 h. The basal enzyme activity in the noninduced cells is approximately 0.3 pmol of resorufin formed per minute per million cells whereas the induced cells show an increase in CYPlAl activity by a factor of 5-7. From the standard control experiment it is unclear whether the method of transfer or the nature of conditions in the recipient flask cause the expression of CYPlAl activity. To determine the cause of this response, several additional experiments were performed. Table 1 demonstrates the effect of varying the method of transferring the cells to a clean spinner flask, to a "conditioned" flask, or to siliconized flasks (or from a siliconized flask), changing the shape of the impeller for either growth or transferring, or using stationary recipient culture methods. For the cases where there is no mixing, in either recipient spinner flask or T-flask, there was almost no change in the response over the basal enzyme activity. In addition, when cells were transferred to a "conditioned" flask, there was no response. Here a "conditioned" flask is a spinner flask previously used to grow microcarrier cultures which is reused as a recipient flask aRer all of the spent liquid medium with microcarriers has been dumped. The method of transferring the cells had no effect on the response relative to the standard control. The effect of agitation rate in the recipient flask on the maximum induction of CYPlAl measured 8 h after initiating transfer is shown in Figure 3. The response was determined at least twice. The stationary spinner flask data from Table 1 represents the 0 rpm point. In the range of 50-150 rpm, there is an almost constant level of CYPlAl expression followed by a dramatic increase, by approximately a factor of 3 when the agitation intensity is increased to 240 rpm. The Kolmogorov-scale eddies can be calculated (see Mufti, 1995) and the maximum CYPlAl activity can be plotted as a function of the ratio of the smallest eddy size to the microcarrier diameter (175pm)(see Figure 4).
a Data represent the average of duplicate samples from a typical experiment. Activity measured 8 h aRer transfer. Represents the average of eight independent experiments. A significant level of p < 0.005 as determined by a two-tailed, unpaired Student's t-test with respect to the corresponding control experiment. h
0
-
PI
0
50
100
150
200
250
300
Agitation Rate (rpm)
Figure 3. Effect of agitation intensity on the maximum induction of CYPlAl in Hep G2 cells in a 50-mL spinner flask. Cells were grown for 3 days at 70 rpm and then transferred to a clean spinner flask and agitated at the designated revolutions per minute for 8 h in the cases of 35,50, 70, and 150 rpm and 2 h for 150,200, and 240 rpm followed by 6 h at 70 rpm. Each point represents the average of duplicate samples.
The effect of increased stress on the induction of CYPlAl is shown in Table 2. Three independent experiments were performed using spinner flasks equipped with a flat-edge baffle to increase the hydrodynamic shear and two independent experiments were performed to demonstrate the effect of depriving the cells of oxygen by transferring the cells to spinner flask with screw caps instead of foam closures. Increasing the hydrodynamic shear increased the CYPlAl expression by a factor of 1.5-2.5 times greater than the standard control. Decreasing the oxygen supply by closing the screw caps on the spinner flasks increased the CYPlAl expression by a factor of 1.4-1.9 times greater than that the standard control. This stress-induced increase in CYPlAl activity must be separated from the effects of chemicals, such as TCDD, that can induce CYPlAl induction in in vitro systems to
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the rest of the liquid medium and medium recirculation. In case 2, where the cells are simultaneously subjected to transfer stress and the full addition of medium and medium recirculation, a single, large response in CYPlAl activity is observed after an 8-h lag. This response was reproducible in a series of such experiments.
Discussion
6
,a
0.2
0.4
0.6
0.8
1
1.2
Ratio of the Eddy Size to the Particle Diameter (rl/d)
Figure 4. Effect of the ratio of eddy size to microcarrier diameter on the maximum induction of CYPlAl in Hep G2 cells in a 50-mL spinner flask. The experimental procedure was as stated for Figure 3. The eddy size was calculated using a kinematic viscosity of 0.01 cm2/s, an impeller diameter of 4 cm, and a power dissipation volume of 40 cm3. Table 2. Effect of Additional Stresses on the Maximum Induction of CYPlAl in Hep G2 Cells Immobilized on Cytodex 3 Microcarriers in 90-mL Spinner Flasks” EROD formation rate (pmol of resorufin formed(lO6 cells/min))b additional percent of stress expt stress control control (%) 1.13 245c increased A 2.77 2.44 186c 4.55 hydrodynamic B 2.44 14oC C 3.42 shea# 1.13 14oC D 1.62 oxygen 185c E 4.06 2.20 deprivation The values are the average of duplicate samples. * Measured activity 8 h afler transfer. Indicates a significance level of p < 0.05 as determined by a two-tailed, unpaired Student’s t-test. Increased hydrodynamic shear was induced by using a modified spinner flask equipped with an additional flat-edged impeller, and an agitation rate of 100 rpm was used.
%
3
E,
v
O 0
5
10
15
20
25
Time (h) Figure 5. Kinetics of CYF’lAl induction in microcarrierattached Hep G2 cells in the two-compartment reactor. In case 1 (denoted by m), the cells are transferred at time t = 0, but into a partially-filled “Liver Compartment” with a free liquidgas interface below the nylon mesh, followed 12 h later with addition of liquid medium to raise the liquid level above the mesh, allowing medium recirculation (see Figure 1B). In case 2 (denoted by O ) , the cells are subjected to transfer to the liver chamber, followed immediately by addition of liquid and medium recirculation. Each point represents the average of duplicate samples for a typical experiment.
probe mechanisms of chemical toxicity. As shown in Figure 5, the method of operation is important to when a low basal level of CYPlAl activity is obtained. In case 1the cells show a modest response to the stress of initial transfer and a second and larger response to addition of
Oxygen deprivation and hydrodynamic shear stress have been shown to increase membrane phsopholipid metabolism (Freyss-Beguin, et al. 1989; Nollert et al., 1989,1991;Bhat and Block, 1992; Michiels et aZ.,1993). In both cases there was a significant increase in prostaglandin synthesis, since the cell models chosen have a disposition for the production of prostaglandins in response to cell stress. We report an additional cellular response to both hydrodynamic shear and oxygen deprivation increase of CYPlAl activity. This response is easily monitored and may be useful in reactor studies as a method to detect stress-induced changes in cell physiolOgY.
Exposure of the human hepatoma cell line, Hep G2, attached to Cytodex 3 microcarriers, to a subtle stress, such as transferring the cells from a conditioned flask to a clean flask resulted in a 5-7-fold increase in the CYPlAl activity (Figure 2). Transfer to a “conditioned flask” rather than a “clean”flask protects the cells from the stress of being transferred and then being exposed to stirring (Table 1). Why “conditioning”of the recipient flask should protect cells is unclear. Washing and autoclaving a “conditioned”flask makes it a “clean”flask, so we suggest that a compound (or compounds) which is formed in the medium due to cellular metabolism must adhere to the glass and must provide a protective effect. Further, siliconization of the spinner flask prior to conditioning does not alter the protective effect of conditioning. Other factors that were examined in the additional control experiments included different methods of transferring the cells. Pouring the cells or pipetting with a normal or detipped pipet had no effect (see Table 1). However, there was a decrease in the response compared to the standard control, if the cells were transferred but then maintained in stationary culture (in either an unconditioned spinner flask or T-flask). Clearly mixing is necessary for CYPlAl activity increases. The results from all of the experiments listed in Table 1indicate that the stress is due to mixing in an unconditioned flask and is independent of the method of transferring the cells. To further investigate the dependence of the inducedCYPlAl activity on hydrodynamic shear stress, we examined the effect of increasing the shear intensity by using both increased agitation rates and a modified spinner flask at higher agitation rates. The increased CYPlAl expression appears to mimic a step-function with respect to agitation (see Figure 3). In order to evaluate the possible mechanism for the induction of CYPlAl as a function of agitation rate, the Kolmogorov theory of isotropic turbulence was applied (Croughan et al., 1987; Cherry and Papoutsakis, 1988; Papoutsakis, 1991). Figure 4 illustrates that CYF’lAl induction occurs at a value of qld less than 1. Below an qld of 0.45, an increase in CYPlAl induction is observed. These results are comparable to the results obtained by Cherry and Papoutsakis (1988), who observed that cell growth decreased linearly with a decrease in qld, starting at an qld = 1.0, with little growth observed below qld = 0.5. Table 2 illustrates that, by increasing the hydrodynamic shear, there is an increase in the inducedCYPlAl activity by 140-245% of the standard control response. Increased hydrodynamic shear was also found
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to occur in a bioreactor designed to examine the biochemical effectof TCDD exposure in the immobilized Hep G2 cells (see Figure 1B). In the reactor it was possible to separate the induction of CYPlAl due to transferring the cells and imposing a stress due to medium recirculation. Figure 5 illustrates the response of the cells if the transferring is immediately followed by initiating medium recirculation or if the medium recirculation is initiated after the level of induced CYPlAl activity due to transferring has subsided. The maximum CYPlAl induction in the first case is equivalent to the sum of the two independent CYPlAl inductions in the latter case. The data are consistent with a mechanism in which increased CYPlAl activity is dependent on hydrodynamic shear stress. The effect of exposing the cells to hypoxia was also examined. Previous in vitro studies have shown that hypoxia resulted in a response similar to that observed for shear stress in endothelial cell lines (Freyss-Beguin et al., 1989; Bhat and Block, 1992; Michiels et al., 1993). Table 2 illustrates that by decreasing the oxygen supply to the cells there was an increase in the induction of CYPlAl over the standard control response. The increase in CYPlAl induction varied from 140 to 185%of the standard control, and it was necessary to transfer the cells to a fresh flask in order to observe the effect of oxygen deprivation. The data presented in this paper demonstrate the ability to use increases in CYPlAl activity as a measure of environmental stresses in the Hep G2 cell line. Although not examined in as much detail, we have observed increases in CYPlAl activity with another human hepatoma, Mz-Hep-1, and a mouse hepatoma, Hepa-1, in response to shear (Mufti et al., 1995) but not in a mutant of Hepa-1 that had a defective Ah receptor. The increase of CYPlAl activity was dependent on the intensity of hydrodynamic shear experienced by the cells and could also be increased by hypoxia. The induction of CYPlAl activity increases in response to environmental stresses may serve as a mechanistic end point for cell stress in cell lines that have retained the ability to induce CYPlAl .
Acknowledgment This work was supported in part by a postgraduate fellowship to N.A.M. by the Natural Sciences and Engineering Research Council of Canada and by a grant from Paracelsian Inc. (Ithaca, NY).The authors would also like to thank Professor Jeff Chalmers and Professor John Babish for their helpful comments.
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