Life Sciences Vol 20, pp . 1735-1740, 1977 . Printed in the U .S .A .

Pergsmon Press

MONOTERTIARYBUTYLHYDROQUINONE EFFECTS ON TETRAHYMENA PYRIFORMIS * John G . Surak Pest . Res . Lab ., Food Science Dept ., Univ . of Florida, Gainesville, FL 32611 (Received in final form April 25, 1977)

Summary The molecular toxicity of monotertiarybutylhydroquinone (TBHQ) was studied using Tetrah mena pyri formis as a model cell system . T a ppm in% t e media inhibited cell jjowth by 50% . 1WBHQ inhibitedthe oxidation of C -acetate to CO In addition, increasing concentrations of TBAQ decreased the incorporation of C-acetate into l ipids and protein, C-1mino acids into protein, H-uridine into RNA ayj H-thymidine into DNA. The incorporation of C-acetate into glycogen increased with concentrations up to 20 ppm TBHQ in the media while glycogen synthesis decreased with 40 ppm TBHQ . Monotertiarybutylhydroquinone (TBHQ) was approved for use as a food antioxidant by the FDA in 1972 (1) . Previous communications indicated that TBHQ was essentially nontoxic to laboratory animals (1, 2, 3) . TBHQ is a microsomal metabolite of another approved antioxidant, butylated hydroxyanisole (BRA) (4) . Allen and Engbloom (5) observed nucleolar fragmentation and the presence of large intranucleolar fibrils in hepatocytes of rhesus monkeys administered BRA at 500 mg/kg body wt for 21 d . When BHA was added to Tetrah mena riformis cultures at 20 ppm there was an inhibition o ce u ar growth and synthesis of DNA, RNA and protein by 50% (6) . This communication reports the action of TBHQ at the cellular level using T . pyriformis as a model cell system . Methods Growth of T . riformis . Tetrah ena riformis strain W were incu ate at t in a medium consisting o 28 proteose peptone and 0 .1% yeast extract . TBHQ as a concentrated solution in dimethyl sulfoxide (DMSO) was added to early log growth cultures, so that the final concentration of TBHQ ranged from 0 ppm to 40 ppm . The concentration of DMSO in the medium was 1 .0% . This level of DMSO does not affect cell growth (6) . Growth of T . pyriformis was measured by counting cell numbers *Florida Agricultural Experiment Station Journal Series No . 348 . 1735

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with a Coulter Counter Model ZBI previously calibrated with polystyrene beads . Radioactive Measurements . The procedures of Shug et al . (7) and ilner as mo i ie y Surak et al . (6) y2re useU-tôdetermine the effec of TBHQ on the incoSpration of C-amino acids }ito protein, H-uridine into RNA, H-thymidine into DNA, and 2Cacetate into lipid, glycogen and protein . A modification of the procedure of Saba and DiLuzio (9) was used to determine the effect of TBHo on the tricar~, xylic acid cycle b measuring the oxidation of "C-acetate to I~CO . TBHQ and 1- I C-acetate were added to a reaction flask (Kontei liatalogue number K-882350) containing 5 ml of culture . The CO 2 was trapped on a 20 mm Whatman 1 filter paper disc saturated with 1 .0 ml hyamine hydroxide . After 0 .5 hr of incubation, the reaction was stopped by the addition of 1 .0 ml concentrated H 2 S0 4 to the reaction flask . Results TBHQ inhibited the growth of Tetrah mena pyriformis during the log growth phase and the finalcell density never reached that of the untreated cultures (Fig . 1) . The inhibition of cellular growth was dose dependent with 26 ppm TBHQ causing a 50% growth inhibition (IC ) . In addition, increasing concentrations of TBHQ in the medium 5q ncreased the generation time of T . pyri formis (Table 1) . TBHQ at 40 ppm decreased the incorporation of 2- 14 C-acetate into protein by 201 (Table 2) . There yjs no significant (P > 0 .05) effect on the incorporation of 2- C-acetate into lipids at TBHQ levels up to 20 ppm whereas concentrations of 40 ppm TBHQ decreased lipid synthesis (P < 0 .01) (Table 1) . Although, 20 ppm TBV~ significantly (P < 0 .05) stimulated the incorporation of 2C-acetate into glycogen (Table 2), a further addition of TBHQ to the medium decreased the synthesis of glycogen (Table 2) . The incorporation of C-amino acids into protein was inhibited by increasing concentrations of TBHQ which became significant (P < 0 .01) at 10 ppm TBHQ (Fig . 2) . The greatest change of rate in the protein synthesis occurred when 10 ppm TBHQ was added to an early log growth culture . A dose dependent reduction in the synthesis of RNA occurred when TBHQ was added to the medium (Fig . 2) . This inhibition became significant (P < 0 .01) when the TBHQ concentrations reached 10 yRm, the shape of the curve being similar to that observed for C-amino acid incorporation into protein (Fig . 2) . A significant decrease (P < 0 .01) occurred in the incorporation of H-thymidine into DNA at 20 ppm TBHQ in the medium (Fig . 2) . At 10 ppm TBHQ in the medium DNA synthesis was reduced by 5% while at 20 ppm TBHQ inhi ted DNA synthesis by 25% . TBHQ inhibited the oxidation of l- C-acetate to CO in a dose dependent manner (Fig . 2) . The shape of the curve wds similar to that observed for protein synthesis (Fig . 2) . Discussion The inhibition of the growth of T . riformis by TBHQ was similar to that reported for BHA (6) . Both antioxidants were dose dependent in inhibiting the generation time, synthesis of macromolecules, and cell growth during the log growth phase (Fig . 1

Vol . 20 . No . 10, 1977

X).

TBHQ on T . pyriformis

1 0

40

10

TIME IN HOURS AFTER AOOITON OF TWO

FIG . 1 Growth curves of T . py rifo~rmis following addition Concenof TBHQ to an early log growth culture . trations in ppm of TBHQ in the media were : ( " ) 0, (0) 10, (A) 20, (O) 40, (o) 60 . TABLE 1 Generation times of T . if is in media containing BHQ Concentration of TBHQ (ppm)

Generation Time (hr)

0

6

10

7

20

10

40 60

is 31

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FIG . 2 Effect of various concentrations of TBHQ on early log growth cultures of T . pyr~i~~fo~rmi~~s . ( " ) CO2 was deterNned after 0 .'Y hrr 1ncuFiat1on with O .I uCi of 1- C-acetate ; ( " ) DNA, ( 11) RNA, and ( O ) protein wers determined after 3 hr incubation with 5 .0 uCi of H-t~Wmidine, 5 .0 uCi of H-uridine, and 0 .5 uCi of C-amino acid hydrosylate respectively . of 26 ppm and 2 ; Table 1 and 2), (6) . However, TBHQ with an IC was somewhat less toxic to T . riformis as compared H BHA with and IC 0 of 20 ppm . The cells a-I e to adapt to the presence of TBHQ iR the medium since the final cell density in the treated cultures never reached that of the untreated cultures (Fig . 1) . A possible major effect of TBHQ on T . pyr~iformi~s~ appears to be c'ä }icreasing the inhibition of the tricarboxylic-acii yc~since concentrations of TBHQ diminished the oxidation of 1- C-acetate to CO (Fig . 2) . In addition, there was a probable subsequent inhibition of electron transport and ATP production . This decrease in cellular energy level could explain the inhibition of macromolecular biosynthesis (Table 2, Fig . 2) . BHA inhibited the synthesis of DNA, RNA, and protein in a similar manner (6) .

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TABLE 2

14 Effect of TBHQ on the incorporation of 2C-acetate into lipids, protein and glycogen of T . pyriformis . Concentration of TBHQ (ppm)

Radioactivity (dpm/10 5 cells t 1 S .D .) In Protein

In Lipid

In Glycogen

0

5513 * 1315

20733 t 3127

278 t 178

10

5375 * 1698

26940 t 5483

332 * 178

20

5453 ± 1728

21062 ± 6348

488 t 216*

40

4397 * 2010

16732 t 4189**

395 ± 187

*P < 0 .05 according to Student paired t-test **P < 0 .01 according to Student paired t-test

The increase in glycogen synthesis at 20 ppm TBHQ can be attributed to an inhibition in protein synthesis and electron transport . Shrago et al . (10) proposed a casual relationship between diminished respiration and protein synthesis with increased gluconeogensis . In addition, cyclohexamide, a drug that inhibits protein synthesis, will stimulate gluconeogensis in T . pyriformis (11) . Since BHA is more lipophilic than TBHQ (12), BHA would be more readily absorbed accounting for the increase in toxicity . This correlates with Milner's work (8) who reported that 0 .136 mM butylated hydroxytoluene (BHT) inhibited cell growth of monkey kidney cells in vitro by 70% while 0 .136 mM BHT-alcohol did not inhibit celluTar growth . The exposure of T . riformis to TBHQ in this series of experiments is possibly greater an the exposure humans would have during estimated maximum consumption (2) . When the medium contained 10 ppm TBHQ, 5x10 5 cells were exposed to 100 ug of antioxidant . This is compared to 1400 to 4900 ug/d, the estimated maximum intake of a 70 ~& man (2) Since a 70 kg man is estimated to contain 1x10 cells (13), it would be highly improbable that concentrations needed to inhibit macromolecular synthesis would occur in vivo . References 1. 2. 3.

B. B. R. 52 _C_ . R.

D . Astill and R. L . Roudabush, Fed . Proc . 32 734a (1973) . D . Astill, C . J . Terhaar, W . J . Krasavage,-U . L . Wolf, L . Roudabush, and D . W . Fassett, J . Am . Oil Chem . Soc . 53-58 (1975) . J . Terhaar, W. J . Krasavage, B . D. Astill, D . W . - Fassett, L . Roudabush, G . L . Wolf, Fed . Proc . 32 223a (1973) .

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4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 .

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A . L . Branen, J . Am . Oil Chem . Soc . 52 59-63 (1975) . J . R . Alen and J . F . Engbloom, Food 'Cosmet . Toxicol . 10 769779 (1972) . J . G . Surak, R . L . Bradley, Jr ., A. L . Branen, and E . Shrago Food Cosmet . Toxicol . 14 277-281 (1976) . A . L . Shug, C . Elson, and E . Shrago, J . Nutr . 99 379-386 (1969) . S . M . Milner, Nature 216 557-560 (1967) . T . M . Saba and N . R. =uzio, J . Lip . Res . 7 566-567 (1966) . E . Shrago, A. L . Shug and S . M. Fergson, Internat . J . Biochem. 2 312-318 (1971) . 'E . Elson, E . Shrago, E . Sondheimer, and M. Yatvin, Biochim . Biophys . Acta 297 125-134 (1973) . 1973 . Eastman Kodak anZ Co ., Publication No . ZG-109 . G . Claus, Clin . Toxicol . 7 497-508 .

Monotertiarybutylhydroquinone effects on Tetrahymena pyriformis.

Life Sciences Vol 20, pp . 1735-1740, 1977 . Printed in the U .S .A . Pergsmon Press MONOTERTIARYBUTYLHYDROQUINONE EFFECTS ON TETRAHYMENA PYRIFORMIS...
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