Evidence of oxidative stress following the viral infection of two lepidopteran insect cell lines.

The infection of Spodoptera frugiperda Sf-9 (Sf-9) and Trichoplusia ni BTI-Tn-5B1-4 (Tn-5B1-4) insect cell lines with Autographa californica multiple nucleopolyhedrovirus (AcMNPV) resulted in increased levels of lipid hydroperoxides and protein carbonyls. In addition, the viral infection resulted in a significant decrease in the reduced glutathione to oxidized glutathione (2GSH/GSSG) ratio. These results are all consistent with an increased level of oxidative stress as a result of the viral infection. It was also observed that the oxidative damage corresponded to reduced cell viability, i.e., the results are consistent with the premise that oxidative damage contributes to cell death. Finally, the measured intracellular activities of most of the antioxidant enzymes, specifically manganese superoxide dismutase (MnSOD), ascorbate peroxidase (APOX), and catalase (CAT, not present in Sf-9 cells), did not significantly decrease following viral infection. In contrast, the measured activity of copper-zinc superoxide dismutase (CuZnSOD) decreased in the Sf-9 and Tn-5B1-4 cells following AcMNPV infection.

Expression of C1 esterase inhibitor by the baculovirus expression vector system: preparation, purification, and characterization.

C1 esterase inhibitor (C1INH) is an important regulator of the classical complement pathway. Hereditary deficiency of C1INH causes angioedema of the skin, gut, and respiratory tissues that may be fatal. C1INH replacement therapy may be lifesaving for patients with this disorder. The objective of this study was to evaluate the use of the baculovirus expression vector system for mass producing biologically active human recombinant (rC1INH). A recombinant baculovirus was constructed coding the human native (nC1INH) sequence under control of the polyhedrin promoter. Spodoptera frugiperda Sf-9 insect cells were infected with this recombinant baculovirus in a medium-scale (10-L) bioreactor to produce rC1INH with a specific activity of 45 U/mg. Purification of rC1INH from the culture harvested at 60 h postinfection yielded 5.9 microg rC1INH/mL supernatant of a 75-kDa product with a specific activity of 31,000 U/mg purified rC1INH compared to 71,000 U/mg purified nC1INH from human serum using the same procedure. This rC1INH was about 25 kDa smaller than nC1INH, suggesting that Sf-9 cells express underglycosylated rC1INH. Glycan analysis showed that both N-glycan and O-glycan chains were present in rC1INH. The N-glycan chains, released using PNGaseF and fluorescently labeled, were analyzed using exoglycosidase treatment and capillary electrophoresis. Their high-mannose structure was consistent with the known failure of the insect cell glycosylation pathway to afford the fully elaborated biantennary structures found on human native nC1INH.

Antioxidant defense systems of two lipidopteran insect cell lines.

Spodoptera frugiperda Sf-9 (Sf-9) and Trichoplusia ni BTI-Tn-5B1-4 (Tn-5B1-4) insect cell lines were found to contain unique assemblages of antioxidant enzymes. Specifically, the Sf-9 insect cell line contained Manganese and Copper-Zinc superoxide dismutase (MnSOD and CuZnSOD) for reducing the superoxide radical (O(2)(*-)) to hydrogen peroxide (H(2)O(2)) and ascorbate peroxidase (APOX) for reducing the resulting H(2)O(2) to H(2)O. Approximately one third of the total SOD activity was found to be MnSOD. The Tn-5B1-4 cells were also found to contain MnSOD (approximately two thirds of the total SOD activity), CuZnSOD and APOX activities. However, the Tn-5B1-4 cell line, in contrast to the Sf-9 cell line, contained catalase (CAT) activity for reducing H(2)O(2) to H(2)O. Both the Sf-9 and Tn-5B1-4 cell lines contained glutathione reductase and dehydroascorbic acid reductase activities for regenerating the reduced forms of glutathione and ascorbic acid, respectively. In addition, both cell lines contained glutathione S-transferase peroxidase activity towards hydroperoxides other than H(2)O(2). Finally, neither cell line contains the glutathione peroxidase activity that is ubiquitous in mammalian cells.

Affinity purification of secreted alkaline phosphatase produced by baculovirus expression vector system.

Human secreted alkaline phosphatase (SEAP) was produced in a stably-transformed Spodoptera frugiperda Sf-9 insect cell line (Sfb4GalT) following infection with a recombinant Autographa californica multiple nuclear polyhedrovirus containing the SEAP gene under control of the polyhedrin promoter. An affinity chromatographic column prepared by linking 4-amino-benzylphosphonic acid to histidyl-expoxy-Sepharose was used to isolate SEAP from the cell supernatant following removal of cells and virus and 10-fold concentration through ultrafiltration. We found that the binding of SEAP on the affinity matrix follows the Langmuir isotherm model. In addition, either recycling SEAP sample through the column for 24 h or loading high SEAP concentrations resulted in a high-purity product. Some nonspecific binding of protein on the matrix occurred when low concentrations of SEAP sample were loaded. Finally, we found that SEAP binding occurs rapidly, i.e., within 30 min of adding the SEAP sample to the affinity matrix.

Separation of alpha-acid glycoprotein glycoforms using affinity-based reversed micellar extraction and separation.

A new method for the preparation of the glycoforms of bovine alpha(1)-acid glycoprotein (AGP) is described relying on affinity-reversed micellar extraction and separation (ARMES). This method has proven effective in separating structurally similar glycoproteins and separating glycoproteins from nonglycosylated proteins from natural sources. In this method, individual glycoforms complex with the lectin, concanavalin A (ConA) are extracted into an organic-phase reversed micellar solution formed by Aerosol OT (AOT). The purity of three AGP glycoforms isolated was assessed by hydroxyapatite high-performance liquid chromatography (HPLC), gel-permeation chromatography and SDS-PAGE. The glycan structure of the pure glycoforms was analyzed. Oligosaccharide mapping using capillary electrophoresis (CE) and PAGE showed the glycans obtained from each glycoform to be distinctly different. ARMES can be used for the semi-preparative scale resolution of the glycoforms of bovine AGP or other therapeutic glycoproteins.

Culture in the rotating-wall vessel affects recombinant protein production capability of two insect cell lines in different manners.

The production of recombinant secreted alkaline phosphatase protein in virally infected insect cells was studied in shaker flask and high aspect rotating-wall vessel (HARV) culture. Two commonly used cell lines, Spodoptera frugiperda Sf-9 (Sf-9) and a nonaggregating isolate of the Trichoplusia ni BTI-Tn-5B1-4 (Tn-5B1-4) cell line, Trichoplusia ni Tn-5B1-4-NA (Tn-5B1-4-NA), were used and monitored for 120-h postinfection. Different responses to culture in the HARV were seen in the two cell lines. While the Sf-9 cell line was able to produce slightly greater amounts of recombinant protein in the HARV than in shaker flask controls, the Tn-5B1-4-NA cell line produced significantly lesser amounts in the HARV than in the shaker flasks. Both cell lines exhibited longer life spans and longer periods of protein production in HARV culture than in shaker flask culture, presumably due to lower levels of shear encountered in the HARV. The important difference was in the protein production rate responses of the two cell lines. While the protein production rates of Sf-9 cells were comparable in both HARV and shaker flask cultures, the protein production rates of Tn-5B1-4-NA cells were much lower in HARV culture than in shaker flask cultures. The conclusion is drawn that cell line-specific adaptation to the HARV strongly influences recombinant protein production.

Recombinant protein synthesis in Trichoplusia ni BTI-Tn-5B1-4 insect cell aggregates.

The Trichoplusia ni BTI-Tn-5B1-4 (Tn-5B1-4) insect cell line has received considerable attention as a host for the baculovirus expression vector system. In the present study, suspension cultures were used to compare Tn-5B1-4 cell aggregates and cells selected to grow predominantly as individual cells. No significant difference was found between cell aggregates and cells growing predominantly individually in regard to cell growth rate, glucose consumption and lactate accumulation, and specific recombinant protein synthesis levels. In addition, the levels of recombinant protein synthesis were considerably higher than those produced by the commonly used Spodoptera frugiperda Sf-9 insect cell line.

Release and preparation of intact and unreduced N-linked oligosaccharides from Sf-9 insect cells.

Glycosylation, the addition of carbohydrates to a peptide backbone, is the most extensive cotranslational and posttranslational modification made to proteins by eukaryotic cells. The glycosylation profile of a recombinant glycoprotein can significantly affect its biological activity, which is particularly important when being used in human therapeutic applications. Therefore, defining glycan structures to ensure consistency of recombinant glycoproteins among different batches is critical. In this study we describe a method to prepare N-linked glycans derived from insect cell glycoproteins for structural analysis by capillary electrophoresis. Briefly, glycoproteins obtained from uninfected Spodoptera frugiperda Sf-9 insect cells were precipitated with ammonium sulfate and the glycans were chemically cleaved by hydrazinolysis. Following the regeneration of the glycan reducing terminal residue and the removal of contaminating proteins and peptides, the glycans were fluorescently labeled by reductive amination. Fluorescent labeling greatly enhanced the detection limit of the glycan structures determined by capillary electrophoresis. Five major glycan structures were found that migrated between tetra-mannosylated hexasaccharide and nonamannosylated undecasaccharide standards. Upon alpha-mannosidase digestion the number of glycan structures was reduced to two major structures with shorter migration times than the undigested glycans. None of the glycans were susceptible to hexosaminidase or galactosidase treatment. These results are consistent with the majority of previous results demonstrating hypermannosylated glycan structures in Sf-9 insect cells.

Electrophoretic analysis of glycoprotein glycans produced by lepidopteran insect cells infected with an immediate early recombinant baculovirus encoding mammalian beta1,4-galactosyltransferase.

Glycosylation, the most extensive co- and post-translational modification of eukaryotic cells, can significantly affect biological activity and is particularly important for recombinant glycoproteins in human therapeutic applications. The baculovirus-insect cell expression system is a popular tool for the expression of heterologous proteins and has an excellent record of producing high levels of biologically active eukaryotic proteins. Insect cells are capable of glycosylation, but their N-glycosylation pathway is truncated in comparison with the pathway of mammalian cells. A previous study demonstrated that an immediate early recombinant baculovirus could be used to extend the insect cell N-glycosylation pathway by contributing bovine beta-1,4 galactosyltransferase (GalT) immediately after infection. Lectin blotting assays indicated that this ectopically expressed enzyme could transfer galactose to an N-linked glycan on a foreign glycoprotein expressed later in infection. In the current study, glycans were isolated from total Sf-9 cell glycoproteins after infection with the immediate early recombinant baculovirus encoding GalT, fluorescently conjugated and analyzed by electrophoresis in combination with exoglycosidase digestion. These direct analyses clearly demonstrated that Sf-9 cells infected with this recombinant baculovirus can synthesize galactosylated N-linked glycans.

Adaptive calibration scheme for quantification of nutrients and byproducts in insect cell bioreactors by near-infrared spectroscopy.

Spectroscopic methods are gaining in popularity in biotechnology because of their ability to deliver rapid, noninvasive measurements of the concentrations of multiple chemical species. Such measurements are particularly necessary for the implementation of control schemes for cell culture bioreactors. One of the major challenges to the development of spectroscopic methods for bioreactor monitoring is the generation of accurate and robust calibration models, particularly because of the inherent variability of biological processes. We have evaluated several methods of building calibration models, including synthetic calibrations and medium spiking methods. The approach that consistently produced reliable models incorporated samples removed from a bioreactor that were subsequently altered so as to increase the sample variation. Several large volume samples were removed from a bioreactor at varying time points and divided into multiple aliquots to which were added random, known amounts of the analytes of interest. Near-infrared spectra of these samples were collected and used to build calibration models. Such models were used to quantify analyte concentrations from independent samples removed from a second bioreactor. Prediction errors for alanine, glucose, glutamine, and leucine were 1.4, 1.0, 1.1, and 0.31 mM, respectively. This adaptive calibration method produces models with less error and less bias than observed with other calibration methods. Somewhat more accurate measurements could be attained with calibrations consisting of a combination of synthetic samples and spiked medium samples, but with an increase in calibration development time.

Simultaneous measurement of glucose and glutamine in insect cell culture media by near infrared spectroscopy.

The purpose of this study was to develop non-invasive techniques to monitor the composition of cell culture media in insect cell bioreactors. Such a monitor could be used in conjunction with a fed-batch feeding scheme to ensure that cells are maintained in an optimal environment for growth and protein production. Glucose and glutamine concentrations in an insect cell culture bioreactor were determined off-line with near-infrared (NIR) absorption spectroscopy. Spectra were collected from 5000 to 4000 cm(-1) with a 1.5-mm optical path length. Partial least squares (PLS) regression was applied to correlate the collected spectra with the concentration of the desired analytes. Under the culture conditions evaluated here, glucose and glutamine concentrations ranged from 38 to 55 mM and from 3 to 13 mM, respectively. Accurate measurements of glucose and glutamine in insect cell culture samples were possible over these entire ranges. The standard error of prediction (SEP) and mean percent error (MPE) for glutamine were 0.52 mM and 5.3%, respectively. Glucose could be measured with an SEP of 1.30 mM and an MPE of 2.3%. These levels of error are quite low considering the changing complexity of the growth media due to the shifting levels of amino acids, carbohydrates, yeastolate, proteins, and cell debris. This study represents an important step in the development of noninvasive on-line monitoring devices for cell culture bioreactors.

Comparison of Trichoplusia ni BTI-Tn-5B1-4 (high five) and Spodoptera frugiperda Sf-9 insect cell line metabolism in suspension cultures.

Nutrient utilization and byproduct accumulation were monitored in Spodoptera frugiperda Sf-9 and Trichoplusia ni BTI-Tn-5B1-4 (High Five) cell lines during growth and following viral infection in suspension cultures in order to develop a better understanding of cell metabolism and to acquire information relevant to large scale fed-batch bioreactors. The utilization of glucose, dissolved oxygen, and amino acids were monitored in Sf-9 cell cultures grown in Sf-900 II serum-free medium (SFM) and in High Five cell cultures grown in both Sf-900 II and Express Five SFM. Using the optimal medium for each cell line, i.e., Sf-900 II SFM for Sf-9 cells and Express Five SFM for High Five cells, the cell growth rate, maximum cell density, specific glucose and glutamine utilization rates, and specific alanine production rate were comparable during cell growth. In addition, the expression level of recombinant human tissue plasminogen activator was comparable in the two cell lines on a per cell basis. It was found, however, that lactate and ammonia accumulated in High Five cell cultures, but not in Sf-9 cell cultures. In addition, High Five cells utilized asparagine more rapidly than glutamine, whereas Sf-9 cells consumed only minimal asparagine, and the oxygen utilization rate was significantly higher in High Five cell cultures. It was also found that the medium had a significant effect on High Five cell metabolism, e.g., the specific glucose utilization rate and the specific lactate and alanine production rates were significantly higher in Sf-900 II SFM than in Express Five SFM. In addition, the maximum cell density and specific asparagine utilization rate were significantly higher in Express Five SFM.

bcl-2 expression in Spodoptera Frugiperda Sf-9 and Trichoplusia Ni BTI-Tn-5B1-4 insect cells: effect on recombinant protein expression and cell viability.

The effect of bcl-2 expression on cell viability and recombinant protein synthesis was investigated in the Spodoptera frugiperda Sf-9 and Trichoplusia ni BTI-Tn-5B1-4 (High Fivetrade mark) insect cell lines. It was found that coinfection with a baculovirus expressing bcl-2 [Autographa californica nuclear polyhedrosis virus (AcNPV)-bcl2] extended the life span of High Fivetrade mark cells but not Sf-9 cells when compared to infection with recombinant baculoviruses expressing either human tissue plasminogen activator (AcNPV-tPA) or Escherichia coli beta-galactosidase (AcNPV-betagal). Similar results were obtained in coinfection experiments; i.e., AcNPV-bcl2 coinfection increased the life span of High Fivetrade mark cells over that of cells infected with either AcNPV-tPA or AcNPV-betagal alone, but they did not affect the life span of coinfected Sf-9 cells. Coinfection of Sf-9 cells with AcNPV-bcl2 and AcNPV-betagal resulted in a decrease in the maximum beta-gal expression levels of over 90% when compared to infection with AcNPV-betagal alone. A similar trend was found in the beta-gal mRNA levels. Coinfection also resulted in a reduced beta-gal expression level in High Fivetrade mark cells, but the reduction was consistent with what would be expected when two recombinant viruses compete for use of the cellular machinery. In contrast to the inhibitory effect of AcNPV-bcl2 coinfection on betagal expression, t-PA expression levels were either not affected (Sf-9 cells) or were increased 50% (High Fivetrade mark cells) over those obtained by infection with AcNPV-tPA alone. These results support the hypotheses that bcl-2 can inhibit transcription of genes under polyhedrin promoter control and that beta-gal expression levels, but not t-PA expression levels, are controlled at the transcriptional level. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 380-390, 1997.

The effect of oscillating dissolved oxygen concentrations on the metabolism of a Spodoptera frugiperda IPLB-Sf21-AE clonal isolate.

The effect of oscillating dissolved oxygen (DO) concentration on the metabolism of a clonal isolate of the Spodoptera frugiperda IPLB-Sf21-AE insect cell line was investigated. Specifically, the effect on cell growth, re- combinant protein synthesis, glucose and glutamine consumption, and lactate accumulation was determined. Prior to conducting the oscillating DO experiments, it was found that the DO concentration could be reduced to 15% air saturation without adversely affecting the growth rate. Under these conditions, glucose and glutamine became depleted as the maximum cell density was reached. The introduction of DO oscillations, that is, cycles consisting of 30 min at 15% DO followed by 30 min of anoxia, significantly altered cell metabolism, including inhibition of cell growth and recombinant protein synthesis. The effect of DO oscillations on glucose consumption was dependent on the experimental conditions. Glucose exhaustion occurred when the DO oscillations contained either an "apparent" anoxia period (nitrogen sparging discontinued upon reaching 0% DO) without pH control or a "true" anoxia period (nitrogen sparging continued throughout anoxia period) with pH control. Glucose consumption was significantly decreased, however, when the cells were exposed to a "true" anoxia period without pH control, that is, low pH inhibited glucose utilization. Glutamine uptake was not significantly affected by DO oscillations. Lactate only accumulated in the oscillating DO runs, a finding consistent with previous results demonstrating that significant lactate accumulation only occurs under DO-limited conditions. (c) 1995 John Wiley & Sons, Inc.

Simultaneous measurement of glucose and glutamine in aqueous solutions by near infrared spectroscopy.

A method is described for measuring the concentrations of both glucose and glutamine in binary mixtures from near infrared (NIR) absorption spectra. Spectra are collected over the range from 5000-4000/cm (2.0-2.5 microns) with a 1-mm optical path length. Glucose absorbance features at 4710, 4400, and 4300/cm and glutamine features at 4700, 4580, and 4390/cm provide the analytical information required for the measurement. Multivariate calibration models are generated by using partial least squares (PLS) regression alone and PLS regression combined with a preprocessing digital Fourier filtering step. The ideal number of PLS factors and spectral range are identified separately for each analyte. In addition, the optimum Fourier filter parameters are established for both compounds. The best overall analytical performance is obtained by combining Fourier filtering and PLS regression. Glucose measurements are established over the concentration range from 1.66-59.91 mM, with a standard error of prediction (SEP) of 0.32 mM and a mean percent error of 1.84%. Glutamine can be measured over the concentration range from 1.10-30.65 mM with a SEP of 0.75 mM and a mean percent error of 6.67%. These results demonstrate the analytical utility of NIR spectroscopy for monitoring glucose and glutamine levels in mammalian and insect cell cultures.

Clonal variation in the Spodoptera frugiperda IPLB-SF21-AE insect cell population.

Clones have been isolated from the heterogeneous Spodoptera frugiperda IPLB-SF21-AE insect cell population. Five of these clones, in addition to the parent cell line and the SF9 cell line (another clonal isolate of the parent cell line), have been compared in regards to morphology, growth, budded virus synthesis, and recombinant protein synthesis. No significant differences in cell morphology were found among these cell lines. There was, however, a significant difference in the average cell size, with diameters ranging from 9.30 +/- 0.184 to 11.11 +/- 0.22 microns and from 9.17 +/- 0.05 to 11.25 +/- 0.24 microns for cells growing in Excell 401 serum-free medium in spinner flask cultures and in TNM-FH medium supplemented with 10% FBS in tissue flask cultures, respectively. While no significant differences in the growth rates were found in TNM-FH medium containing 10% calf serum, significant differences were found in Excell 401 serum-free medium, with population doubling times ranging from 38.5 +/- 6.6 to 64.5 +/- 6.4 h in spinner flask studies. Significant differences in expression levels of Escherichia coli beta-galactosidase (beta-gal) were also found in both 12-well plates and spinner flasks. In the 12-well plate studies, the peak levels of beta-galactosidase obtained by these cell lines ranged from 0.332 +/- 0.091 to 0.805 +/- 0.117 mg/10(6) cells and from 0.580 +/- 0.130 to 1.458 +/- 0.132 mg/10(6) cells in Excell 401 and Hyclone Hy-Q serum-free media, respectively. In the spinner flask studies, peak expression levels ranged from 0.128 +/- 0.053 to 0.573 +/- 0.215 mg/10(6) cells in Excell 401 serum-free medium.(ABSTRACT TRUNCATED AT 250 WORDS)

Review and patents and literature. The use of insect cell cultures for recombinant protein synthesis: Engineering aspects.

The use of the insect cell/baculovirus expression system for producing recombinant proteins of bacterial, plant, insect, and mammalian origin has become widespread. The popularity of this eukaryotic expression system is due to many factors, including (1) potentially high protein expression levels, (2) ease and speed of genetic engineering, (3) ability to accommodate large DNA inserts, (4) protein processing similar to higher eukaryotic cells (e.g., mammalia cells), and (5) ease of insect cell growth (e.g., suspension growth). The following review of the literature discusses two engineering aspects of recombinant protein synthesis by insect cell cultures: bioreactor scale-up and insect cell line selection. Following this review patent abstracts and additional literature pertaining to expression of recombinant proteins in insect cell culture are listed.

Sparged animal cell bioreactors: mechanism of cell damage and Pluronic F-68 protection.

Pluronic F-68 is a widely used protective agent in sparged animal cell bioreactors. In this study, the attachment-independent Spodoptera frugiperda Sf9 insect cell line was used to explore the mechanism of this protective effect and the nature of cell damage in sparged bioreactors. First, bubble incorporation via cavitation or vortexing was induced by increasing the agitation rate in a surface-aerated bioreactor; insect cells were rapidly killed under these conditions of the absence of polyols. Supplementing the medium with 0.2% (w/v) Pluronic F-68, however, fully protected the cells. Next, cell growth was compared in two airlift bioreactors with similar geometry but different sparger design; one of these bioreactors consisted of a thin membrane distributor, while the other consisted of a porous stainless steel distributor. The flow rates and bubble sizes were comparable in the two bioreactors. Supplementing the medium with 0.2% (w/v) Pluronic F-68 provided full protection to cells growing in the bioreactor with the membrane distributor but provided essentially no protection in the bioreactor with the stainless steel distributor. These results strongly suggest that cell damage can occur in the vicinity of the gas distributor. In addition, these results demonstrate that bubble size and gas flow rate are not the only important considerations of cell damage in sparged bioreactors. A model of cell death in sparged bioreactors is presented.

Structural features of nonionic polyglycol polymer molecules responsible for the protective effect in sparged animal cell bioreactors.

The nonionic surfactant Pluronic F-68 polyol is commonly used to protect cultured animal cells from the detrimental effects of sparging. In this study we investigated the structural features of the Pluronic F-68 molecule responsible for this protective behavior. Poly(oxyethylene)-poly(oxypropylene) block copolymer polyols of various molecular weights and percentages of hydrophobe (poly(oxypropylene], including both Pluronic and reverse Pluronic polyols, were considered. The potential toxicity of these agents was examined in the absence of sparging (i.e., in spinner flasks) by using the attachment-independent Sf9 insect cell line as a model system. Each polyol resulted in one of three distinct types of behavior in these spinner flask experiments: (1) cells lysed at an exponential rate, (2) inhibition of cell growth (i.e., no net cell growth), or (3) uninhibited cell growth. It was then shown that all of the Pluronic and reverse Pluronic polyols that did not inhibit cell growth provided protection from sparging in the bioreactors used in this study; thus, finding a polyol that protected cells was synonymous with finding one that did not inhibit cell growth. The ability of these polyols to protect animal cells in sparged bioreactors was found to correlate well with the hydrophilic-lipophilic balance (HLB). Those polyols with the largest HLB values were found to be protective agents. These poly(oxyethylene)-poly(oxypropylene) polyols were also shown to be more effective protective agents than pure poly(oxyethylene); thus, the presence of the hydrophobe (poly(oxypropylene] is important in their ability to serve as protective agents.