Toxicology Letters, 28 (1985) 93-98 Elsevier

93

TOXLett. 1479

PYRUVATE AND RELATED c~-KETOACIDS PROTECT MAMMALIAN

CELLS IN CULTURE AGAINST HYDROGEN PEROXIDE-INDUCED

CYTOTOXICITY

(Antioxidants; Hz02 toxicity in vitro; pyruvate; V79 Chinese hamster cells)

ULRICH ANDRAE, JASWANTH SINGH* and KYRIAKOULA ZIEGLER-SKYLAKAKIS

Gesellschafi fiir Strahlen- und Urnweltforschung (GSF), Abteilung fiir Toxikologie, D-8042

Neuherberg/Miinchen (F. R. G.)

(Received May 28th, 1985)

(Revision received August ‘19th. 1985)

(Accepted August 22nd, 1985)

SUMMARY

Pyruvate efficiently protected V79 Chinese hamster cells against the lethal effects of hydrogen perox-

ide. Protection was also provided by other a-ketoacids, such as a-ketobutyrate, a-ketoglutarate and 01.

ketoadipate, although higher concentrations were required. The corresponding P-ketoacids had no ef-

fect. The results indicate that pyruvate and other cu-ketoacids possess antioxidant activity in vitro and,

probably, in viva.

INTRODUCTION

Mammalian cells in vivo are permanently exposed to hydrogen peroxide (H202)

and other reactive forms of oxygen which are formed during numerous enzymatic

reactions [ 1,2]. Most of these oxygen species are toxic [3], potentially mutagenic

[4,5], and carcinogenic [6]. Cells have developed a variety of defense strategies

against the deleterious effects of oxygen radicals. In addition to several enzymatic

detoxication mechanisms [7], a number of biological antioxidants exist, such as

ascorbate, glutathione, oc-tocopherol, p-carotene, and uric acid, which provide pro-

tection against oxygen toxicity. We now present evidence that cu-ketoacids, abun-

Abbreviations: BME, Basal medium Earle; DMEM, Dulbecco’s modified Eagle’s medium; MEM,

Eagle’s minimal essential medium; FCS, fetal calf serum; HBSS, Hank’s balanced salt solution.

*Permanent address: CSIR, Regional Research Laboratory, Jammu Tawi (India)

0378.4274/85/$ 03.30 (3 Elsevier Science Publishers B.V.

94

dantly present in mammalian cells, constitute another, not previously recognized,

group of antioxidants.

MATERlALS AND METHODS

Chemicals Sodium pyruvate and other ketoacids were purchased from Sigma, Miinchen

(F.R.G.); Hz02 (Perhydrol, suprapur) from Merck, Darmstadt (F.R.G.); cell

culture media and antibiotics from Biochrom, Berlin (F.R.G.); FCS from Gibco,

Karlsruhe (F.R.G.).

Cell culture V79 Chinese hamster cells were grown in 90-mm Falcon dishes at 37°C in a

humidified atmosphere of 95% sir/5% CO*. The growth medium consisted of

Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FCS, 100

U penicillin/ml and 100 pg streptomycin/ml.

Toxicity of Hz02 300 V79 cells were seeded into 90-mm Falcon Petri dishes containing 10 ml of

basal medium Earle (BME) supplemented with 10% fetal calf serum (FCS), 100 U

penicillin/ml and 100 fig streptomycin/ml (complete BME) and allowed to attach

for 4 h at 37°C. Cells were then washed with 5 ml Hank’s balanced salt solution

(HBSS), replenished with fresh BME without serum and exposed to various Hz02

concentrations in the presence or absence of sodium pyruvate for 3 h. After treat-

ment, cells were washed twice with 5 ml HBSS and incubated with complete BME.

After one week of undisturbed growth, the plates were rinsed with HBSS, fixed in

methanol, stained with hematoxylin, and counted.

RESULTS AND DISCUSSION

The toxicity of Hz02 for V79 Chinese hamster cells, incubated with or without

sodium pyruvate in the culture medium, is shown in Fig. 1. Hz02 was very toxic in

the absence of pyruvate, killing 98% of the cells at 25 PM. The survival curve was

shouldered, indicating the existence of a saturable repair process or a detoxication

mechanism operating with a limited capacity. Toxicity was greatly reduced in the

presence of 70 yM pyruvate. The survival curve had a much broader shoulder with

virtually complete survival of the cells at 12.5 PM H202. At this concentration,

Hz02 killed more than 50% of the cells in the absence of pyruvate. Treatment with

25 PM Hz02 in the presence of 70 PM pyruvate was tolerated by 60% of the cells.

In the presence of 1 mM pyruvate, cells were completely protected even from 50 PM

HzO2, the highest concentration tested, which killed more than 99.9% of the cells

when no pyruvate was present.

95

Fig. I. Effect of pyruvate on the toxicity of H202 to V79 Chinese hamster cells. 300 cells in BME were exposed to H2Oz in the presence or absence of sodium pyruvate for 3 h. The absolute plating efficiency of the control cells not treated with H2Oz or pyruvate was 48 T 16%. Survival of treated cells is nor- malized to the plating efficiency of the respective control. Each point represents the mean of 4 indepen- dent experiments (each on triplicate plates) t SD. 0, Hz02; A, Hz02 + 70 $4 pyruvate; 0, Hz02 + 1 mM pyruvate.

We examined whether other naturally occurring cr-ketoacids would also protect V79 cells. The results shown in Table I indicate that this was the case. Complete pro- tection against the cytotoxic effects of 25 pM T-I202 was provided by 1 mM CY- ketobutyrate, a-ketoglutarate, or ol-ketoadipate. In contrast, acetoacetate, ~3- ketoglutarate, or p-ketoadipate were ineffective, suggesting that the capacity to pre- vent HzOz-induced toxicity is a property of N-, but not P-ketoacids.

At a lower concentration (70 FM) pyruvate was considerably more effective than the other cz-ketoacids in protecting cells against the lethal effects of 25 PM Hz02 (Table I). Only 10% of the cells survived when ~-ketobutyrate, ~-ketoglutarate or cu-ketoadipate were present, whereas approximately 60% did so in the presence of pyruvate.

The antioxidant property of pyruvate and the other a-ketoacids is most likely the

96

TABLE I

EFFECTS OF 01-

Hz02

Treatment

None

Pyruvate

a-Ketobutyrate

a-Ketoglutarate

wKetoadipate

AND /%KETOAClDS ON THE SURVIVAL OF V79 CELLS EXPOSED TO 25 pM

Percent relative survival

~ Hz02 + Hz02 _

100 2* 1

(1 mM) 104 i 3 99% 1

(1 mM) 99 + 1 99* 2

(1 mW 99 k 2 99* 2

(1 mM) 98 i 1 97* 1

Acetoacetate (1 mM) 95 i 3 5i 5

P-Ketoglutarate (1 mM) 99 f 3 5* 4

,3-Ketoadipate (1 mM) 95 + 6 5+ 5

Pyruvate (70 rM) 95 * 4 61 t 18

w-Ketobutyrate (70 I’M) 98 k 1 10 * 10

a-Ketoglutarate (70 ILM) 99 t 2 9k 8

wKetoadipate (70 I’M) 99 i 1 II + 10

Results represent the mean I SD of 3 independent experiments, each on triplicate plates.

consequence of a chemical reaction with Hz02 leading to their oxidative decarbox-

ylation with concomitant decomposition of Hz02 [8]. This mechanism is supported

by recent experiments by us showing a rapid destruction of Hz01 in culture media

containing pyruvate. Changes in Hz02 concentration were followed spec-

trophotometrically at 240 nm (data not shown).

Sodium pyruvate, at concentrations from l-5 mM, is a normal constituent of

several, but not all, commonly used cell culture media. Previously, only little atten-

tion has been paid to this component. In the light of the present findings, however,

several results concerning the effects of Hz02 on mammalian cells in vitro must be

reinterpreted. This applies not only to investigations on the action of Hz02 itself but

also to studies of other effects in which Hz02 has been implicated. For example, ir-

radiation of aqueous solutions of riboflavin, tryptophan and tyrosine with visible

fluorescent light yields very high levels of Hz02 [9]. When these medium com-

ponents, at identical concentrations, are irradiated in DMEM containing 1 mM

pyruvate, the amount of Hz02 formed is much lower [9]. Todd et al. [lo] found that

marsupial cells were killed when exposed to blacklight radiation in MEM. When the

culture medium was changed to Ham’s F-12, which contains pyruvate, killing was

reduced to lo-50%. Similar apparent contradictions concerning the toxicity [ 1 l] or

lack of toxicity [ 121 to mammalian cells of media preirradiated with fluorescent light

might similarly be resolved when the presence or absence of pyruvate in the different

cell culture media used is considered.

The fact that pyruvate has a strong antioxidant activity should also be taken into

consideration in the development of cell culture media. Pyruvate could, for exam-

ple, be chosen to replace catalase in certain media [13].

97

In addition to its importance in the detoxification of Hz02 in vitro, the antioxi- dant activity of pyruvate and other a-ketoacids deserves atttention for another reason. The concentration of pyruvate in the serum of adult humans is about 70 PM [14], a concentration high enough to efficiently protect cells against Hz02 in vitro (Fig. 1). Therefore, significant antioxidant activity may be provided by the presence of pyruvate in human blood. Blood concentrations of other cu-ketoacids are much lower than that of pyruvate [15], e.g., approx. 10pM for ol-ketoglutarate [16]. Thus, these compounds are unlikely to be involved in the detoxification of H202 in blood. In several organs, however, concentrations of a-ketoacids are much higher. In rat liver, where the concentration of pyruvate is about 500 PM [17], the concentration of a-ketoglutarate amounts to about 220 PM [18]. Therefore, in organs such as the liver, ol-ketoacids other than pyruvate may also play a significant role in preventing HzOz-induced toxicity.

ACKNOWLEDGEMENT

The excellent technical assistance of Ms. Heike Homfeldt is gratefully acknowl- edged.

REFERENCES

1 I. Fridovich, Superoxide dismutases, Ann. Rev. Biochem., 44 (1975) 147-159.

2 B. Chance, H. Sies and A. Boveris, Hydroperoxide metabolism in mammalian organs, Physiol. Rev.,

59 (1979) 527-605.

3 B. Halliwell and J.M.C. Gutteridge, Oxygen toxicity, oxygen radicals, transition metals and disease,

Biochem. J., 219 (1984) 1-14.

4 C.S. Moody and H.M. Hassan, Mutagenicity of oxygen free radicals, Proc. Natl. Acad. Sci. USA,

79 (1982) 2855-2859.

5 M.L. Cunningham and B.R. Lokesh, Superoxide anion generated by potassium superoxide is

cytotoxic and mutagenic to Chinese hamster ovary cells, Mutat. Res., 121 (1983) 299-304.

6 A. Ito, H. Watanabe, M. Naito and Y. Naito, Induction of duodenal tumors in mice by oral ad-

ministration of hydrogen peroxide, Cann 72 (1981) 174-175.

7 H.M. Hassan and 1. Fridovich, Superoxide dismutases: detoxication of a free radical, in W.B. Jakoby

(Ed.) Enzymatic Basis of Detoxication, Vol. I, Academic Press, New York, 1980, pp. 31 l-332.

8 C.A. Bunton, Oxidation of a-diketones and cu-keto-acids by hydrogen peroxide, Nature, 163 (1949)

444.

9 R.J. Wang and B.T. Nixon, Identification of hydrogen peroxide as a photoproduct toxic to human

cells in tissue-culture medium irradiated with ‘daylight’ fluorescent light, In Vitro, 14 (1978) 715-722.

10 P. Todd, C.B. Schroy and M.R. Lebed, Post-irradiation effects of photoreactivating light and caf-

feine on cultured marsupial cells exposed to ultraviolet light, Photochem. Photobiol., 18 (1973)

433-436.

1 I J.D. Stoien and R.J. Wang, Effect of near-ultraviolet and visible light on mammalian cells in culture,

II. Formation of toxic photoproducts in tissue culture medium by blacklight, Proc. Natl. Acad. Sci.

USA, 71 (1974) 3961-3965.

12 M.O. Bradley and N. Sharkey, Mutagenicity and toxicity of visible fluorescent light to cultured mam-

malian cells, Nature, 266 (1977) 724-726.

98

13 F.J. Darfler and P.A. Insel, Clonal growth of lymphoid cells in serum-free media requires elimination

of Hz02 toxicity, .I. Cell. Physiol., 115 (1983) 31-36.

14 A. Tsirimbas and W. Stich, ijber die Brenztraubensaure im Blut und Serum bei malignen Tumoren

des Menschen, Klin. Wschr., 38 (1960) 1196-1198.

I5 Wissenschaftliche Tabellen (Documenta Geigy), 7th ed., Ciba-Geigy, Basel, 1973, p. 603.

16 H. Kaser, Zur Technik der papierchromatographischen Trennung und Bestimmung der ol-Ketosauren

im Blute, Clin. Chim. Acta, 6 (1961) 337-346.

17 H. Sies, C. Gerstenecker, K.-H. Summer, H. Menzel and L. FlohC, Glutathione-dependent

hydroperoxide metabolism and associated metabolic transitions in hemoglobin-free perfused rat liver,

in L. Flohe, H.C. Benohr, H. Sies, H.D. Wailer and A. Wendel (Eds.), Glutathione, Thieme, Stutt-

gart, 1974, pp. 261-276.

18 E.A. Siess, D.G. Brocks and O.H. Wieland, Distribution of metabolites between the cytosolic and

mitochondrial compartments of hepatocytes isolated from fed rats, Hoppe-Seyler’s Z. Physiol.

Chem., 359 (1978) 785-798.