Pluronic Enhances the Robustness and Reduces the Cell Attachment of Mammalian Cells

The addition of the non-ionic surfactant, Pluronic F-68, to serum-free CHO cultures causes multi-functional effects that enhance cell yield in agitated cultures and reduce cell adhesion in stationary cultures. Three independent CHO cell lines were subjected to high liquid shear in assay systems that either included or excluded a liquid-gas interface. In the absence of Pluronic, there was a loss in cell viability in either assay system, although there was an intrinsic variability in sensitivity of the cell lines to shear damage. Supplementation with Pluronic prevented loss of cell viability, indicating protection in either a gas sparged or bubble-free environment. However, we found no evidence of long-term protection of cells once Pluronic was removed. Pluronic was capable of repairing trypsin-damaged cells as evidenced by enhanced growth, reduced membrane porosity, and improved robustness under liquid shear. The proportion of adherent cells was reduced to a minimal level by the presence of Pluronic although its effect was rapidly reversible with a high proportion (70%) of adherent cells observed within a few culture passages of its removal. The observed effects of Pluronic on these cultures are compatible with a mechanism in which the polymer forms a protective layer on the cell membrane, which has a significantly lower hydrophobicity.     

Interactions between animal cells and gas bubbles: The influence of serum and pluronic F68 on the physical properties of the bubble surface

We describe a method by which the degree of bubble saturation can be determined by measuring the velocity of single bubbles at different heights from the bubble source in pure water containing increasing concentrations of surfactants. The highest rising velocities were measured in pure water. Addition of surfactants caused a concentration-dependent and height-dependent decrease in bubble velocity; thus, bubbles are covered with surfactants as they rise, and the distance traveled until saturation is reached decreases with increased concentration of surfactant. Pluronic F68 is a potent effector of bubble saturation, 500 times more active than serum. At Pluronic F68 concentrations of 0.1% (w/v), bubbles are saturated essentially at their source. The effect of bubble saturation on the interactions between animal cells and gas bubbles was investigated by using light microscopy and a micromanipulator. In the absence of surfactants, bubbles had a killing effect on cells; hybridoma cells and Chinese hamster ovary (CHO) cells were ruptured when coming into contact with a bubble. Bubbles only partially covered by surfactants adsorbed the cells. The adsorbed cells were not damaged and they also could survive subsequent detachment. Saturated bubbles, on the other hand, did not show any interactions with cells. It is concluded that the protective effect of serum and Pluronic F68 in sparged cultivation systems is based on covering the medium-bubble interface with surfaceactive components and that cell death occurs either after contact of cells with an uncovered bubble or by adsorption of cells through partially saturated bubbles and subsequent transport of cells into the foam region. (c) 1994 John Wiley & Sons, Inc.     

Insights into protective effects of medium additives on animal cells under fluid stresses: the hydrophobic interactions

Animal cells in suspension culture can suffer severe mechanical damage from bursting gas bubbles or other hydrodynamic force sources. Certain chemical additives in the culture media, particularly some surface-active chemicals, can effectively protect animal cells against such damage. Previously we proposed that the protective effect is associated with the adsorption of the additives in the cell membrane through hydrophobic binding of the surface-active molecules to the membrane. Adsorption of the additives to the cell membrane may lead to decreased hydrophobicity of the cell surface, thus eliminating cell adhesion to bubbles and reducing cell damage from bursting bubbles. In this study, we measured the hydrophobicity of two insect cell lines based on cell adhesion to hydrocarbon phase and its influence by surface-active chemicals, Pluronic F68, a methylcellulose and a polyethylene glycol. The experimental results showed strong support for the aforecited cell protection mechanism.     

The fate of Pluronic F-68 in chondrocytes and CHO cells

The surfactant Pluronic F-68 (PF-68) is widely used in large-scale mammalian cell culture to protect cells from shear stress that arises from agitation and gas sparging. Several studies suggested that PF-68 is incorporated into the cell plasma membrane and could enter the cells, but without providing any direct evidence. The current study has examined this question for two cell types, one of pharmaceutical interest (CHO cells) and the other of biomedical interest (chondrocytes or cartilage cells). A fluorescent derivative of PF-68 was synthesized to detect and localize internalized Pluronic with culture time. PF-68 uptake by the cells was quantified and characterized. We clearly demonstrate that PF-68 enters the cells, and possibly accumulates in the endocytic pathway. CHO cells showed an average uptake of 11.7 +/- 6.7 (SEM) microg PF-68/10(6) cells while the uptake of chondrocytes was 56.0 +/- 10.9 (SEM) microg PF-68/10(6) cells, independently of the initial PF-68 concentration (between 0.01 and 0.2%, w/v) and of cell concentration (from 1 x 10(6) to 4 x 10(6) cells/mL). These uptake values were identical for both static and agitated culture conditions. Finally, we found that CHO cells are able to eliminate intracellular fluorescent PF-68 but chondrocytes are not. These results show that the uptake of PF-68 by the cells can severely affect PF-68 concentration in the culture medium and thus shear protection effect.     

Analysis of cell-to-bubble attachment in sparged bioreactors in the presence of cell-protecting additives

To investigate the mechanisms of cell protection provided by medium additives against animal cell injury in sparged bioreactors, we have analyzed the effect of various additives on the cell-to-bubble attachment process using CHO cells in suspension. Cell-to-bubble attachment was examined using three experimental techniques: (1) cell-bubble induction time analysis (cell-to-bubble attachment times); (2) forming thin liquid films and observing the movement and location of cells in the thin films; and (3) foam flotation experiments. The induction times we measured for the various additives are as follows: no additive (50 to 500 ms), polyvinyl pyrrolidone (PVP: 20 to 500 ms), polyethylene glycol (PEG: 200 to 1000 ms), 3% serum (500 to 1000 ms), polyvinyl alcohol (PVA: 2 to 10 s), Pluronic F68 (5 to 20 s), and Methocel (20 to 60 s). In the thin film formation experiments, cells in medium with either F68, PVA, or Methocel quickly flowed out of draining thin liquid films and entered the plateau border. When using media with no additive or with serum, the flow of cells out of the thin liquid film and film drainage were slower than for media containing Pluronic F68. PVA, or Methocel. With PVP and PEG, the thin film drainage was much slower and cells remained trapped in the film. For the foam flotation experiments, a separation factor (ratio of cell concentration in the foam catch to that in the bubble column) was determined for the various additives. In the order of increasing separation factors (i.e., increasing cell attachment to bubbles), the additives are as follows: Methocel, PVA, Pluronic F68, 3% serum, serum-free medium with no additives, PEG, and PVP. Based on the results of these three different cell-to-bubble attachment experiments, we have classified the cell-protecting additives into three groups: (1) Pluronic F68, PVA, and Methocel (reduced cell-to-bubble attachment); (2) PEG and PVP (high or increased cell-to-bubble attachment); and (3) FBS (reduced cell attachment butslower drainage films compared with F68, PVA, and Methocel with some cell entrapment in those films). These phenomena are discussed in relation to the interfacial properties of the media reported in a companion Study (this issue). (c) 1995 John Wiley & Sons Inc.     

A polystyrene microsphere assay for detecting surface hydrophobicity variations within Candida albicans populations

Adherence of microbial pathogens to host cell surfaces may involve hydrophobic interactions. Here, we describe the development of an assay for detecting cell surface hydrophobicity of populations and individual cells of the opportunistic fungal pathogen Candida albicans. The assay involves mixing polystyrene latex microspheres with cells and subsequent enumeration of cell-attached microspheres. Similar levels of hydrophobicity within a population of yeast cells were obtained with the microsphere assay and with a commonly used aqueous-hydrocarbon biphasic partitioning assay. Various buffers were found to support detection of surface hydrophobicity with the microsphere assay. Complex fungal growth media did not. Serum in test media prevented microsphere attachment. A unique advantage of the assay compared to others is that individual cells can be assessed for surface hydrophobicity. Within a population of C. albicans yeast cells, strongly, moderately and weakly hydrophobic cells were observed. Within some pairs of mother-daughter cells, only one cell was hydrophobic. Germ tbes and hyphae were hydrophobic regardless of the hydrophobic status of the parent cell. These results indicate that the microsphere assay is a useful test evaluating cell surface hydrophobicity of C. albicans.     

Quantitative investigations of cell-bubble interactions using a foam fractionation technique

Previous work by the authors and others has shown that suspended animal cell damage in bioreactors is caused by cell-bubble interactions, regardless whether the bubbles are from bubble entrainment or direct gas sparging. As approach to measure the adsorptivity of animal cells to bubbles, a modified batch foam fractionation technique has been developed in this work and proven to be applicable. By using this technique, the number of cells adsorbed per unit bubble surface area and the adsorption coefficients have been measured to quantify hybridoma cell-bubble interactions, and the prevetive effects of serum and Pluronic F68 on these interactions. It was demonstrated quantitatively that the hybridoma cells adhere to bubbles spontaneously and significant numbers exist in the foam, and that both the serum and Pluronic F68 provide strong prevention to these cell-bubble interactions. The results obtained provide criteria for bioreactor operation and medium formulation to prevent cell-bubble interactions and cell damage in the culture processes.Abbreviations NBCS new born calf serum - SFM serum-free medium     

Mechanical agitation of hybridoma suspension cultures: Metabolic effects of serum, pluronic F68, and albumin supplements

Metabolic effects of the medium supplements, fetal bovine serum (FBS), Pluronic F68, and bovine serum albumin (BSA) were compared for agitated bioreactor cultures of hybridoma cells. Agitation speeds up to 600 rpm, without entrainment of gas bubbles by sparging or vortex formation, allowed examination of cell interactions with turbulent fluid forces. For cultures in FBS-supplemented RPMI media, there was no significant effect of intense turbulent fluid shear on cell growth, metabolism, or antibody, production. Serum-free cultures (Pluronic F68 or BSA supplements) at 600 rpm demonstrated greatly increased glycolysis rates during exponential growth relative to controls. Nutrient limitations caused increased rates of decline of the viable cell concentrations and a reduction in final antibody titers by around 70%. The Pluronic F68 and BSA supplements did not lead to cell protection by modifying metabolism under conditions of intense turbulent fluid shear. Supplementing the protein-free medium with FBS reduced glycolysis rates in exponential growth phase, but this did not prevent a high rate of viable cell decline and low antibody titers. We concluded that FBS does not have a metabolic effect on cells subjected to intense turbulent fluid shear. Although the agitation conditions employed in this study were more intense than generally required for agitated bioreactor culture of hybridomas, we have demonstrated the importance of considering metabolic effects of turbulent fluid forces on cultures using nutrient-rich basal media, in addition to the considerations of gas bubble effects described by other workers. (c) 1992 John Wiley & Sons, Inc.     

Poly-γ-glutamic acid enhances the growth and viability of Chinese hamster ovary cells in serum-free medium

The protective effects of polymer additives, including a group of viscosity-enhancing polymer poly-γ-glutamic acid (γPGA; 10, 50, and 500?kDa) and surface-active polymer Pluronic F68, on Chinese hamster ovary cells against damage due to shear stress were investigated in shake-flask cultures. The level of protection was dependent upon the molecular weight of γPGA and its concentration. When 0.05 or 0.075?% of 500?kDa γPGA was added, the cell growth and viability were almost equal to those of Pluronic F68 supplementation and were much higher than those of the control without additives. For the first time, we show that γPGA is another environmentally-friendly medium additive that can be used in place of Pluronic F68.     

Interfacial properties of cell culture media with cell-protecting additives

In an effort to identify key rheological properties that contribute to cell protection against shear damage, we have measured surface shear and dilatationai viscosities, dynamic surface tension, foaminess, and foam stability for media containing cell-protecting additives. In a companion article,(18) we found that cell-to-bubble attachment was decreased in media containing Methocel, Pluronic F68, or polyvinyl alcohol (PVA). In medium containing polyethylene glycol (PEG) or potyvinyl-pyrrolidone (PVP), attachment was increased. PEG, PVP, serum (FBS), and serum albumin (BSA) increased the surface viscosity of the air/medium surface (thus, producing a more rigid interface), whereas F68 and PVA lowered it greatly. Foaming experiments showed that Methocel, PEG, PVA, and F68 decreased the foam half-life while FBS, BSA, and PVP were foam stabilizers. Interestingly, the foam stability of CHO cell suspensions decreased significantly for cell concentrations higher than ca. 2 x 10(6) cells/mL. Nonviable CHO cells reduced foam stability further. Dynamic surface tension values of the media tested were found significantly differentfrom their static surface tension values. The interfacial properties measured and the results presented in the companion study suggest that the additives that lower dynamic surface tension the most (Methocel, F68, and PVA) correlate well with reduced cell-to-bubble attachment, and thus, cell protection. Reduced dynamic surface tension with these additives implies faster surfactant adsorption, mobile interfaces, lower surface viscosity, and foam destabilization. Because PEG and PVP resulted in increased cell-to-bubble attachment and had different interfacial properties, a different mechanism (compared with Methocel, PVP, and F68) is apparently responsible for their protective effect. Finally, cell protection offered by FBS and BSA is attributed to the foam stabilization properties provided by these additives. (c) 1995 John Wiley & Sons Inc.     

Effect of surfactant pluronic F-68 on CHO cell growth, metabolism, production, and glycosylation of human recombinant IFN-γ in mild operating conditions

The control of glycosylation to satisfy regulatory requirements and quality consistency of recombinant proteins produced by different processes has become an important issue. With two N-glycosylation sites, γ-interferon (IFN-γ) can be seen as a prototype of a recombinant therapeutic glycoprotein for this purpose. The effect of the nonionic surfactant Pluronic F-68 (PF-68) on cell growth and death was investigated, as well as production and glycosylation of recombinant IFN-γ produced by a CHO cell line that was maintained in a rich protein-free medium in the absence or presence of low agitation. Under these conditions, a dose-dependent effect of PF-68 (0-0.1%) was shown not only to significantly enhance growth but also to reduce cell lysis. Interestingly, supplementing the culture medium with PF-68 led to increased IFN-γ production as a result of both higher cell densities and a higher specific production rate of IFN-γ. If cells were grown with agitation, lack of PF-68 in the culture medium decreased the fraction of the fully glycosylated IFN-γ glycoform (2N) from 80% to 65-70% during the initial period. This effect appeared to be due to a lag phase in cell growth observed during this period. Finally, a global kinetic study of CHO cell metabolism indicated higher efficiency in the utilization of the two major carbon substrates when cultures were supplemented with PF-68. Therefore, these results highlight the importance of understanding how media surfactant can affect cell growth as well as cell death and the product quality of a recombinant glycoprotein expressed in CHO cell cultures.     

A low-cost chemically defined protein free medium for a recombinant CHO cell line producing prothrombin

A chemically defined protein free medium, DF6S, was developed for the cultivation of a recombinant Chinese hamster ovary cell line (CHO2DS) producing human prothrombin in suspension batch culture. DF6S was formulated by optimizing DME/F12 with amino acids and supplementing the optimized DME/F12 with aurintricarboxylic acid, ethanolamine, ferric sulfate, Pluronic F68, putrescine and sodium pyruvate. From a seeding density of 2.3 × 10⁵ cells ml^–1, CHO2DS cells grown in suspension in DF6S medium reached a maximal cell density of 1.92 × 10⁶ cells ml^–1 with an accumulated prothrombin concentration of 16.7 mg l^–1 after 6 days in culture.     

Pluronic F68-Linoleic Acid Nano-spheres Mediated Delivery of Gambogic Acid for Cancer Therapy

Gambogic acid (GA), a natural compound from gamboge resin, has been introduced as a promising antitumor drug contributing to its broad spectrum of antitumor activity. However, the poor aqueous solubility and short half-life hinder its clinical application. Pluronic F68 (F68) is a well-known amphiphilic block copolymer consisting of hydrophobic propylene oxide units and hydrophilic ethylene oxide. Although F68 has an amphiphilic structure, its short propylene oxide segment limits its dilution stability and drug-loading capacity. To overcome this limitation, we modified F68 by conjugating linoleic acid, a hydrophobic fatty acid, to increase the hydrophilic-hydrophobic interaction and thus improve the stability of F68 nano-spheres. This F68-linoleic acid (F68-LA) conjugate was synthesized and was used to load GA to improve its anticancer effects. GA-loaded F68-LA nano-spheres were stable for 6 days, with a mean diameter of 159.3 nm and zeta potential of ?23.2 mV. The entrapment efficiency of GA in F68-LA nano-spheres was as high as 92.0%. Furthermore, F68-LA/GA nano-spheres exhibited an enhanced cytotoxic activity and proapoptotic effect against human ovarian cancer A2780 cells, compared with free GA. Our results showed that the F68-LA/GA nano-spheres might be a promising cancer-targeted drug delivery system in ovarian cancer therapy.     

Modulating and optimizing Pluronic F-68 concentrations and feeding for intensified perfusion Chinese hamster ovary cell cultures

Perfusion culture is often performed with micro-sparger to fulfill the high oxygen demand from the densified cells. Protective additive Pluronic F-68 (PF-68) is widely used to mitigate the adverse effect in cell viability from micro-sparging. In this study, different PF-68 retention ratio in alternating tangential filtration (ATF) columns was found to be crucial for cell performance of different perfusion culture modes. The PF-68 in the perfusion medium was found retained inside the bioreactor when exchanged through ATF hollow fibers with a small pore size (50 kD). The accumulated PF-68 could provide sufficient protection for cells under micro-sparging. On the other hand, with large-pore-size (0.2 μm) hollow fibers, PF-68 could pass through the ATF filtration membranes with little retention, and consequently led to compromised cell growth. To overcome the defect, a PF-68 feeding strategy was designed and successfully verified on promoting cell growth with different Chinese hamster ovary (CHO) cell lines. With PF-68 feeding, enhancements were observed in both viable cell densities (20%–30%) and productivity (~30%). A threshold PF-68 concentration of 5 g/L for high-density cell culture (up to 100 × 10⁶ cells/mL) was also proposed and verified. The additional PF-68 feeding was not observed to affect product qualities. By designing the PF-68 concentration of perfusion medium to or higher than the threshold level, a similar cell growth enhancement was also achieved. This study systematically investigated the protecting role of PF-68 in intensified CHO cell cultures, shedding a light on the optimization of perfusion cultures through the control of protective additives.     

Hydrophobicity of Bacillus and Clostridium spores.

The hydrophobicities of spores and vegetative cells of several species of the genera Bacillus and Clostridium were measured by using the bacterial adherence to hexadecane assay and hydrophobic interaction chromatography. Although spore hydrophobicity varied among species and strains, the spores of each organism were more hydrophobic than the vegetative cells. The relative hydrophobicities determined by the two methods generally agreed. Sporulation media and conditions appeared to have little effect on spore hydrophobicity. However, exposure of spore suspensions to heat treatment caused a considerable increase in spore hydrophobicity. The hydrophobic nature of Bacillus and Clostridium spores suggests that hydrophobic interactions may play a role in the adhesion of these spores to surfaces.     

Hydrophobicity of Bacillus and Clostridium spores

The hydrophobicities of spores and vegetative cells of several species of the genera Bacillus and Clostridium were measured by using the bacterial adherence to hexadecane assay and hydrophobic interaction chromatography. Although spore hydrophobicity varied among species and strains, the spores of each organism were more hydrophobic than the vegetative cells. The relative hydrophobicities determined by the two methods generally agreed. Sporulation media and conditions appeared to have little effect on spore hydrophobicity. However, exposure of spore suspensions to heat treatment caused a considerable increase in spore hydrophobicity. The hydrophobic nature of Bacillus and Clostridium spores suggests that hydrophobic interactions may play a role in the adhesion of these spores to surfaces.     

Effect of shear stress on expression of a recombinant protein by Chinese hamster ovary cells

A flow chamber was used to impart a steady laminar shear stress on a recombinant Chinese hamster ovary (CHO) cell line expressing human growth hormone (hGH). The cells were subjected to shear stress ranging from 0.005 to 0.80 N m(-2). The effect of shear stress on the cell specific glucose uptake, cell specific hGH, and lactate productivity rates were calculated. No morphological changes to the cells were observed over the range of shear stresses examined. When the cells were subjected to 0.10 N m(-2) shear in protein-free media without Pluronic F-68, recombinant protein production ceased with no change in cell morphology, whereas control cultures were expressing hGH at 0.35 microg/10(6 )cells/h. Upon addition of the shear protectants, Pluronic F-68 (0.2% [w/v]) or fetal bovine serum (1.0% [v/v] FBS), the productivity of the cells was restored. The effect of increasing shear stress on the cells in protein-free medium containing Pluronic F-68 was also investigated. Cell specific metabolic rates were calculated for cells under shear stress and for no-shear control cultures performed in parallel, with shear stress rates expressed as a percentage of those obtained for control cultures. Upon increasing shear from 0.005 to 0.80 N m(-2), the cell specific hGH productivity decreased from 100% at 0.005 N m(-2) to 49% at 0.80 N m(-2) relative to the no-shear control. A concurrent increase in the glucose uptake rate from 115% at 0.01 N m(-2) to 142% at 0.80 N m(-2), and decreased lactate productivity from 92% to 50%, revealed a change in the yield of products from glucose compared with the static control. It was shown that shear stress, at sublytic levels in medium containing Pluronic F-68, could decrease hGH specific productivity.     

CHO cell growth and recombinant interferon-γ production: Effects of BSA, Pluronic and lipids

The role of bovine serum albumin in mammalian cell cultures and the possibility of its substitution by other components in a serum-free medium has been investigated. In this study, BSA was shown to be important for growth and product formation in CHO cells expressing recombinant human interferon-. There were indications that its stimulating growth effect was dependent on the source of BSA used and probably was related to the purification procedure used for the production of the desired albumin fraction. Cell growth did not occur in the absence of BSA but at low concentration (1 mg ml^–1) it was stimulated by the addition of a combination of a commercial lipid mixture plus Pluronic F68. However, under the latter conditions IFN- production was adversely effected. The importance of individual lipid components was investigated using a statistical approach based on a Plackett-Burman design. Linoleic acid was identified as a positive variable for cell growth while cholesterol was identified as a negative variable for both cell growth and IFN- production. When a combination of linoleic acid plus Pluronic F68 was included in the formulation of low BSA medium, cell growth was similar to that at high BSA concentration (5 mg ml^–1) but the IFN- concentration was significantly reduced (ca. 45%).Abbreviations IFN- interferon- - CHO Chinese Hamster Ovary cells - BSA bovine serum albumin - FAF-BSA fatty acid-free bovine serum albumin     

Differential cell surface properties of vegetative Bacillus

Aims:  The genus Bacillus encompasses a wide range of species which display varying pathogenic abilities. The hydrophobicity of a range of Bacillus species was determined to evaluate the correlation between bacterial hydrophobicity and pathogenicity.
Methods and Results:  Bacterial adhesion to hydrocarbon assays were used to determine the hydrophobicity of various Bacillus species. Significant differences in the hydrophobicity of vegetative Bacilli were found. Specifically, vegetative Bacillus anthracis or Bacillus thuringiensis cells were highly hydrophobic whereas Bacillus cereus or Bacillus subtilis were only slightly hydrophobic using this test. Cell adhesion assays using A549 or J774 cells were used to demonstrate a correlation between the bacterial hydrophobicity profiles with the ability to adhere to the mammalian cell lines.
Conclusions:  The ability of Bacillus species to adhere to mammalian cell lines correlates with the hydrophobicity of the bacteria and also correlates with the relative pathogenicity of some of the Bacillus species tested.
Significance and Impact of the Study:  This work suggests that study of the physical-chemical properties of vegetative cells could inform future approaches for the rapid identification and discrimination of potentially pathogenic Bacilli .     

Comparative genotoxic effects of the cooked-food-related mutagens Trp-P-2 and IQ in bacteria and cultured mammalian cells

As part of a major study to evaluate the mutagenicity of chemicals produced during the cooking of foods, we examined the responses of bacteria and cultured Chinese hamster cells to the compounds Trp-P-2 (3-amino-1-methyl-5H-pyrido[4,3-b]indole) and IQ (2-amino-3-methylimidazo[4,5-f]quinoline), constituents identified in cooked beef and fish. In the Ames/Salmonella tester strain TA1538, both compounds were confirmed to be extremely potent mutagens that were active at levels below 1 ng/plate in the presence of hamster-liver S9 microsomal fraction. 50-fold higher doses of both compounds were required for mutagenicity in the uvr+ tester strain TA1978. Trp-P-2 also behaved as a strong mutagen in CHO cells using the standard exogenous activation with hamster-liver S9 fraction. At concentrations below 1 microgram/ml it produced dose-dependent increases in cell killing, mutations at the hprt and aprt loci, sister-chromatid exchanges, and chromosomal aberrations. An excision-repair-deficient strain was about 2-fold more sensitive than the normal CHO cells with respect to these genotoxic effects of Trp-P-2. IQ had unexpectedly weak activity for all genetic endpoints in the CHO cells, and it produced clear-cut responses only in the repair-deficient cells and only above a concentration of 10 micrograms/ml. The toxicity that was observed with IQ was not affected by the repair capacity of the cells and was not associated with chromosomal aberrations, indicating that damage to cellular structures other than nuclear DNA was likely the predominant pathway for cell killing. Because the culture conditions normally used for CHO cell exposure were shown to be competent in producing bacterial mutagenicity with IQ, it was concluded that the active metabolite of IQ was present in the medium but was somehow ineffective in reaching the DNA of CHO cells and/or reacting with it. These results suggest that the relative mutagenic potency of compounds in Salmonella may bear no direct relationship to relative mutagenicity in CHO cells, emphasizing precaution in attempting to extrapolate microbial data to mammalian somatic cells. This study illustrates the use and merits of a multi-endpoint assay for genetic damage in CHO cells, the utility of using CHO cells that are defective in excision repair of DNA, and the importance of comparative testing between bacterial and mammalian systems.