in vitro Cell. Dev. Biol.--Animal 31:876-879, December 1995 © 1995 Society for In Vitro Biology 1071-2690/95 $05,00+0.00

PRODUCTION OF ETHANOL BY CULTURED INSECT CELLS

MASAKAZU TAKAHASHI, YOSHIAKI KONO, KAZUHIRO MATSUSHITA. AND JUN MITSUHASHI

National Institute of Health, Shinjuku-ku, Tok+o 162, Japan ( M. T., Y. K.); Saitama Medical School, Moroyama, Saitama 350-04, Japan (K. M.); and Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183, Japan (J. M.)

(Received 3 March 1995; accepted 26 June 1995)

SUMMARY

Proton Nuclear Magnetic Resonance (tH-NMR) Analysis of insect cell culture media used for cultivating insect cell lines derived from the Heshfly Sarcophaga peregrina, swalIowtaiI butterfly PapiIio xuthus, and cabbage armyworm Mamestra brassicae revealed that ethanol appeared in the medium as the cultures aged. By incorporating p3C-1]-glucose into the media, we pursued JzC-NMR spectrograms to show that the ethanol was derived from glucose. Thus, it became evident that the insect cells cultured in vitro produce ethanol from glucose as a metabolite.

Key words: insect cells; ethanol production; insect cell metabolite; NMR-analysis; glucose metabolism.

INTRODUCTION

Many insect cell lines have been established during the last three decades. Recently. their use in various fields of science and tech- nology has increased. However, basic studies on cultured insect cells themselves, especially metabolic products of insect cells, are scarce. During a study in which Nuclear Magnetic Resonance (NMR) anal- yses were made of the media used for culturing insect cells, we noticed the presence of ethanol at considerable concentrations. In the present study, we verified that the ethanol was produced by insect ceils using glucose as a carbon source.

MATERIALS AND METHODS

Cell lines. The N1H-SaPe-4 cell line derived from whole embryos of the fleshfly Sarcophaga peregrina (13), the NIAS-PX-64 cell line derived from the pupal ovaries of lhe swallowtail butterfly Papilio xuthu,~ (6), and the NIAS- MaBr-92 cell line derived from larval hemoeytes of the cabbage armyworm Mamestra brassicae (I 1), were used.

Culture media. All of the cell lines were maintained in the Mitsuhashi Maramorosch (MM) medium containing 3% fetal bovine serum (10). In some cases, one-fourth of the glucose was replaced with [J3C-1]-glucose (99% atom%; Isotec lnc., Miamisburg, OH) so that its metabolic products could be detected by lzC-NMR.

Cultivation. The fleshfly cell line was cultured for 6 d, and the other two cell lines for 8 d. All of the experimental cultures were maintained in triplicate at 25 ° C.

Sampling. Cells and the media used for the cultures were harvested on Days 3 and 6 for the fleshfly cell line, and Days 4 and 8 for butterfly and armyworm cell lines. During sampling, cell density was measured with a Thoma's hemacytometer. We centrifuged the culture media at 400 × g for 10 rain to remove the cells. The media were stored at - 20 ° C until analyzed.

NMR annly~es. Each sample was further centrifuged at 7000 X g at 4 ° C f~r 20 min. The supernatant was filtered through a molecule sieve (Moleut T; UFPI TGC 24; Nihon Millipore Kogyo, Tokyo, Japan), which excluded mol- ecules with a molecular weight of more than 10 000. An aliquot of 450/al of the supernatant (pH 6.5-7.0) was placed in an NMR tube (5 mm outer di- ameter) with 50 pl of 10 mM 3-trimethylsilyl 2,2,3,3-tetradeutropropionic

876

acid (TSP) in D20 (Isotec Inc., Miamisburg, OH). The D20 served as an internal lock for the magnetic field. The TSP and dioxane were used as stan- dards for ~H- and 13C- chemical shifts, respectively. The NMR spectra were measured at room temperature with a JEOL-JNM EX 400 FT-NMR spec- trometer. Assignment of JH- and r~C-signals was made by comparison to standard chemicals. We estimated concentrations of metabolic products by comparing the signal intensity in NMR analysis to signal intensities of cor- responding standard compounds of known concentration.

RESULTS

Cell growth. Cell density increased steadily with time in all cul- tures (Fig. 1).

Detection of components of the medium by ~H-NMR. The ~H-NMR spectrogram of fresh MM medium and that of the medium used for cultivating armyworm cells for 4 d are shown in Fig. 2. In the spec- trograms, leucine [chemical shift: 1.004 ppm, doublet peaks (d)], isoleueine (1.022, triplet peaks (t)], valine (1.054, t), lactate (1.327, d), u-alanine (1.473, d), acetate [1.924, singlet peak, (s)], pyruvate (2.377, s), succinate (2.413, s), glycine (3.565, s) and glucose (4.638, d) were assigned by our checking chemical shifts and peak profiles of each chemical. These components were also detected in the media used for cultivating butterfly and fleshfly cells, but the change in concentration of these components was only slightly inconsistent dur- ing cell growth.

The armyworm celt medium also displayed triplet signals of eth- anol not found in fresh medium (Fig. 2). Ethanol was also observed in the media used for cultivating the fleshfly and butterfly cells. Con- centrations of ethanol in each medium used for the cultures were calculated based on the standard substance TSP and by comparing the peak height of tH-NMR spectrograms (1.187, t) with that of the ethanol standard (10 nrM) (Table 1). In the fleshfly and butterfly cell lines, the concentration of ethanol increased from 0 to 15.41 and 18.46 mM, respectively, during culture. In the armyworm cell line,

ETHANOL PRODUCTION BY INSECT CELLS 877

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Days of culture

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NIAS-PX-64 4.77 _+ 1.12 8.46 + 2.59 (butterfly)

NIAS-MaBr-92 11.15 + 4.56 2.75 + 3.91 (armyworm)

in various insect cell cultures before (2,3,7,8), these substances were not considered further in this study. The clear and consistent change noticed in the ~H-NMR analysis was the appearance of ethanol in the media used for culturing cells. In Fig. 2, only a typical spectro- gram is shown; however, ethanol was detected in all of the media used for culturing cells for 3 or 4 d or more (Table 1). Therefore, we assumed that cultured insect cells produced ethanol as a metabolite.

In the next step, we examined how the ethanol was produced. After substituting one-quarter of the glucose incorporated into the MM medium with [~3C-1]-glucose, the media used for culturing ceils were analyzed with HC-NMR. We found that the concentration of ethanol

FIG. 1. Growth curves of three cell lines at 25 ° C.

the concentration was the highest on the fourth day of culture and then decreased. Differences in cell density and growth rate between cultures makes comparisons of their ethanol production difficult to interpret.

Starting material for production of ethanol. Because the MM me- dium contained glucose as a sole carbohydrate, we assumed that the ethanol was produced from glucose. To prove this assumption, we used glucose whose C-1 position was substituted with ~sC isotope and analyzed the medium used for culture with ~3C-NMR. When the cells were cultured in the medium containing [~3C-1]-glucose, a sig- nal of the methyl moiety of ethanol (19.1) appeared in the medium cultured for 3 or 4 d in all of the cell lines tested (Figs. 3-5). Cell growth was accompanied by an increase in the methyl signal and a decrease of [C-1] signals of ct-pyranose and 13-pyranose (94.8, 98.7), indicating a gradual production of labeled ethanol. In the medium for each cell line cultured for 6 or 8 d, the ethyl signal of lactate (22.9) was obvious. Other signals of carbons included in ethanol (59.8) and lactate (71.1) structures were not detected by ~3C-NMR analysis.

DISCUSSION

In the ~H-NMR spectrogram, several amino acids and some other organic acids were detected in addition to glucose and ethanol. How- ever, the concentration of these acids did not show consistent changes with age of the cultures. Because changes in amino acid and some other organic acid concentrations in media have been studied

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FIG. 2. 1H-NMR spectrograms of fresh MM medium (upper) and the MM medium used for cuhivation of armyworm cells for 4 d (lower). Ace, acetate; tt-Ala, ct-alanine: Ethanol, ethanol; Gly, glycine; Glu, glucose; Ile, isoleucine; Lac, lactate; Leu, leueine: Pyr, pyruvate; Suc, succinate; TSP, 3-trimethylsilyl 2,2,3,3-tetradeutrupropionic acid; Val, valine.

878 TAKAHASHI ET AL.

increased and that of glucose decreased with age of the culture (Figs. 3-5), suggesting that a part of the glucose was converted to ethanol. When cells were cultured for a longer period, lactate appeared in the media. This probably indicates that a part of the ethanol was further converted to lactate. Examining the ~:~C-NMR spectrograms, we found that the peaks of ethanol and lactate were derived solely from [':~C-1]-glucose.' The ethanol and lactate were not produced spontaneously, because they were not detected when the fresh me- dium alone was incubated.

There have been several reports about the effects of ethanol on insect ceils. Mitsuhashi et al.(9) reported that the growth of Antheraea eucalypti cells and Aedes aegypti cells was slightly stimulated by 0.5% ethanol, whereas it was impaired by 2.0% ethanol and the cells were killed within a short period by 4.0% ethanol. In Drosophila cell cultures, cell multiplication was stimulated by addition of ethanol to the culture medium at a concentration of 1-2.5%, whereas it was impaired by 5% ethanol (4). In organ cultures, evagination and dif- ferentiation of Drosophila imaginal discs were promoted by 0.5% ethanol (12), and the development of ovaries of Tenebrio molitor was

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promoted by 0.1-0.15% ethanol (5). Ashburner (1) reported that the most suitable medium for Drosophila salivary gland cultures was Grace's medium containing ethanol at the final concentration of 8.0%. No other effects of ethanol on cultured insect cells or organs have been reported. The maximum concentration of ethanol observed in the present study was 11.5 mM ( = 0.053%). The physiological significance of ethanol at this concentration was obscure.

The foregoing experiments showed that insect cells produced eth- anol as a metabolite. It may be true for most, if not all, cultured insect cells, because the cells used in the present study were derived from distantly related insect species and furthermore, from different tissues.

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FIG. 3. Changes in the peaks of glucose and ethanol in the MM medium containing [~sC-l_]-glucose during cultivation of the fleshfly cell line (13C- NMR spectrograms). Upper: fresh MM medium; middle: after 3 d; lower: after 6 d. Dioxane (1 n'd//) is the standard substance. Each unit on the ppm scale equals 1 ppm.

REFERENCES

1. Ashburner, M. Patterns of puffing activity in the salivary gland chro- mosomes of Drosophila. Introduction by ecdysone in salivary glands of D. melanogaster cultured in vitro. Chromosoma 38:255-281; 1972.

2. Bedard, C.; Tom. R.; Kamen, A. Growth, nutrient consumption, and end- product accumulation in Sf-9 and BTI-EAA insect cell cultures: in- sights into growth limitation and metabolism. Bioteehnol. Prog. 9:615~24; 1993.

3. Chao, J.; Ball, G. H. A comparison of amino acid utilization by cell lines of Culex tarsalis and Culex pipiens. In: Kurstak, E.; Maramorosch, K., eds. Invertebrate tissue culture, applications in medicine, biology, and agriculture. New York: Academic Press; 1976:263-266.

ETHANOL PRODUCTION BY INSECT CELLS 8 7 9

~-pyranose C-1

(l-pyranose C-1

DIoxana

Ethanol

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4. Horikawa, M.; Ling, J.-N. L.; Fox, A. S. Effects of substrates on gene- controlled enzyme activities in cultured embryonic cells of Drosoph- ila. Genetics 55:569-583; 1967.

5. Laverdure, A.-M. Culture in vitro de l'ovaire nymphale de Tenebrio mol- itor (Coleoptere) en presence d'ecdysone. C.R. Acad. Sci. D-269:82- 85; 1969.

6. Mitsuhashi, J. Establishment of cell lines from the pupal ovaries of the swallow tail, Papilio xuthus Linne (Lepidoptera, Papilionidae). Appl. Entomol. Zool. 8:64-72; 1973.

7. Mitsuhashi, J. Determination of essential amino acids for insect cell lines. In: Maramorosch, K.; Mitsuhashi, J., eds. Invertebrate cell cul- ture applications. New York: Academic Press; 1982:9-51.

8. Mitsubashi, J. Differences in amino acid metabolism among ceil lines derived from cabbage armyworm, Mamestra brassicae (Lepidoptera, Noctuidae). Appl. Entomol. Zool. 22:533-536; 1987.

9. Mitsuhashi, J.; Grace, T. D. C.: Waterhouse, D. F. Effects of insecticides on cultures of insect cells. Entomol. Exp. Appl. 13:327-341; 1970.

10. Mitsuhashi, J.; Maramorosch, K. Leafhopper tissue culture: embryonic, nymphal, and imaginal tissues from aseptic insects. Contrib. Boyce Thompson Inst. 22:435-469; 1964.

11. Mitsuhashi, J.; Shozawa, A. Continuous cell lines from larval hemocytes of the cabbage armyworm, Mamestra brassicae. Develop. Growth Dif- fer. 27:5994506; 1985.

12. Sengel, P.; Manderon, P. Aspects morphologiques du developpement in vitro des disques imaginaux de la Drosophil. C.R. Acad. Sci. D- 268:405407; 1969.

13. Takahashi, M.; Mitsuhashi, J.; Ohtaki, T. Establishment of a cell line from embryonic tissues of the fleshfly, Sarcophaga peregrina (Insecta, Diptera). Develop. Growth Differ. 22:11-19: 1980.

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FIG. 5. Changes in the peaks of glucose and ethanol in the MM medium containing [13C-l]-glueose during cultivation of the butterfly cell line (~3C- NMR spectrograms). Upper: after 4 d; lower: after 8 d. Dioxane (1 mM) is the standard substance. Each unit on the ppm scale equals 1 ppm.