Inhibition of lactate dehydrogenase in cultured SIRC cells by cigarette smoke

Histochemistry 55, 307-317 (1978) I-Iistochemistry �9 by Springer-Verlag 1978

Inhibition of Lactate Dehydrogenase in Cultured SIRC Cells by Cigarette Smoke*

Norman Fleming**, Heinrich Walt, and Gonzague S. Kistler Division of Cell Biology, Institute of Anatomy, University of Zfirich, Gloriastrai3e 19, CH-8006 Ziirich, Switzerland

Summary. SIRC cell monolayer cultures were exposed to whole smoke from a mid tar and nicotine level research cigarette (ASFC, 72 puffs), or from a high tar and nicotine level reference cigarette (Kentucky 2R1, 48 puffs) over a period of 65 days. The activity and distribution of lactate dehydrogenase (LDH) in the cells were investigated, and the electrophoretic characteristics of its isozymes studied. Cell morphology was examined by light microscopy and by transmission- and scanning electron microscopy.

L D H activity was reduced by exposure to smoke from both cigarette types, the greater inhibitory effect being produced by that of the Kentucky cigarette. In addition, cells exposed to this high tar and nicotine smoke displayed intramitochondrial granules which were larger and more numerous than those found in cells exposed to the mid tar and nicotine smoke, or in the control cells. It is speculated that cation accumulation in the mitochondria may be involved in the observed inhibition of L D H activity.

Introduction

Cultured cells exposed to cigarette smoke, smoke condensate, or several specific components of smoke have been found to display biochemical and morphological alterations. Thus, variations in D N A content and synthesis, altered growth characteristics, and modified ultrastructure were observed in various cell lines of human and rodent origin after exposure to whole cigarette smoke or to its gas vapour phase (Leuchtenberger et al., 1973 a, 1973 b, 1974; Leuchtenberger and Leuchtenberger, 1974; Holt et al., 1975; Davies et al., 1975; Styles, 1976). Malignant t ransformation has also been induced in cultured cells by the action

* Supported by a research grant from the ASFC (Association Suisse des Fabricants de Cigarettes), Switzerland ** Present address: Department of Pathology, The Research Institute, The Hospital for Sick Children, 555, University Avenue, Toronto, Ontario, Canada, M5G l x 8 Send offprint requests to G.S. Kistler, MD, Division of Cell Biology, Institute of Anatomy, University of Ziirich, Gloriastr. 19, CH-8006 Zfirich.

0301-5564/78/0055/0307/02.20

308 N. Fleming et al.

of cigarette smoke, some of its subfractions, or chemicals (e.g. polyaromatic hydrocarbons) that are known constituents of smoke (Di Paolo et al., 1971; Inui and Takayama, 1971; Rhim and Huebner, 1973; Leuchtenberger and Leuchtenberger, 1974; Benedict et al., 1975; Davies et al., 1975; Evans and Di Paolo, 1975).

Not all cell lines, however, react in the same way to smoke exposure. In earlier investigations (Fleming and Kistler, 1977) we were unable to detect ultrastructural changes in cultured Veto (monkey kidney) cells after 260 puffs of smoke from a mid tar, mid nicotine (ASFC) research cigarette. The histochemical and electrophoretic characteristics of several enzymes also remained similar to those of the controls, and inocula of cell suspensions failed to produce tumours in nude mice. We speculated that this apparent resistance to change might have been the result of an earlier, non-malignant transformation of the Vero cell line to a very stable condition.

In the present study, cultured cells of another well-established line, SIRC, were exposed to smoke from the same ASFC cigarettes (mid tar and nicotine) or to smoke from the Kentucky reference cigarette (high tar and nicotine) over a 65 day period. Variations in the activity and distribution of lactate dehydrogenase (LDH) were investigated, and the electrophoretic mobility of its isozymes studied. Cell morphology was examined at the light microscope level, and by transmission-and scanning electron microscopy.

Materials and Methods

Cell Culture

The SIRC cells (Staatens Seruminstitut Rabbit Cornea, Oryctolagus coniculus Flow Laboratories, Irvine, Scotland) were obtained in passage 444. They were grown in 35 mm plastic culture dishes in a 95% air :5% CO2 atmosphere at 37~ and 100% relative humidity. The culture medium consisted of Eagle's Minimum Essential Medium (MEM) containing Earle's Balanced Salt Solution (BSS, Serva TC Feinbiochemica, Heidelberg, West Germany) with the following additional components per 100 ml: 10 ml foetal bovine serum (reduced to 4 ml in maintenance medium); 30 mg glutamine; 10,000 IU penicillin and 10 mg streptomycin sulphate. The medium was buffered to pH 7.3 7.4 with 12% NaHCO3 and sterilized by Millipore filtration (0.22 g). The cell cultures were divided into five groups: 1) untreated negative controls; 2) exposed to smoke from ASFC research cigarettes, 3) sham exposed to the same number of puffs of air as puffs of smoke in group 2; 4) exposed to smoke from Kentucky 2R1 reference cigarettes; 5) exposed to the same number of puffs of air as puffs of smoke in group 4. The cultures were exposed to two (Kentucky) or three (ASFC) puffs of cigarette smoke daily from Monday to Thursday of each week, and subcultured (1:3) on Fridays. For exposure, the medium was removed from the cultures and replaced with enough fresh medium to just cover the cells. The monolayers were then exposed to 10% cigarette smoke in 95% a i r+5% CO2 as described by Davies and Kistler (1974). Standard CORESTA conditions were observed, i.e. puff volume 35 ml, duration 2 sec, frequence 1 per rain. The cells were exposed to 72 puffs of smoke from the ASFC cigarette or to 48 puffs from the Kentucky cigarette over a total period of 65 days (9 passages).

Cigarettes

The two types of research cigarettes used were a) the ASFC (Association Suisse des Fabricants de Cigarettes) cigarette (total particulate matter 18.9 rag, nicotine 1.16 mg per cigarette) which was tipped with a 20 mm cellulose acetate filter; b) the Kentucky 2R1 reference cigarette (Tobacco

Inhibition of Lactate Dehydrogenase by Cigarette Smoke 309

and Health Research Institute, University of Kentucky, Lexington, Kentucky, USA; total particulate matter 28.8 mg and nicotine 2.19 mg per cigarette), which was used without a filter.

Cytochemistry

The cells were fixed in situ (10 min, 4 ~ C) in freshly prepared 4% paraformaldehyde in 0.1 M potassium phosphate buffer (pH 7.4). The fixative was rinsed off with 0.9% NaCI and the cells were stained for L D H (30 min, 37 ~ C) in a solution containing the following components per 100 ml: 1M Na-DL-Iactate, pH 7.0, 10 ml; nitroblue tetrazolium (NBT), 35 rag; NAD, 66 rag; phenazine methosuiphate (PMS), 4 rag; 0.5 M Tris/HCl pH 7.0, 10 ml; distilled water, 80ml. Con- trols were incubated without substrate and were always negative.

Polyacrylamide Gel Eleetrophoresis

For each group of cultures, cells f iom two petri dishes were transferred to a 75 cmz culture flask and allowed to reach confluency (3 days). The cells were detached from the flasks by the addition to the medium of Na~EDTA, final concentration 0.005 M and the suspension (approx. 4 x 107 cells) was centrifuged at 800 rpm for 10 rain. The pellet was washed with 0.9% NaC1 and centrifuged again. The cells were resuspended in 0.15 ml 0.05% aqueous sodium cholate and disrupted by three freezing/thawing cycles in liquid nitrogen. The suspension was centrifuged at 10,000 g for 20 rain, the supernatant removed and mixed with an equal volume of 40% sucrose solution. Twenty gl aliquots of this mixture were subjected to electrophoresis at pH 8.8 in 75 x 5 m m tubes of 8% polyacrylamide gel by the method of Maizel (1971) for high pH discontinuous systems. A current of 3 m A m p per tube was applied for 90 rain at 4 ~ C. The gels were transferred to test tubes and the isozymes of L DH visualized by staining in the same solution as that used for the cytochemical demonstrat ion of the enzyme.

Enzyme Assay

The cell supernatant prepared for gel electrophoresis was diluted 1 : l0 with 0.9% NaCI and L D H was assayed by the method of Bergmeyer and Bernt (1974). Protein was determined by the method of Lowry et al. (1951), and enzyme activity expressed as units per g protein.

Electron Microscopy

a) Transmission EM: The cells were fixed in situ for 20 rain in an ice cold solution of 2.5% glutaraldehyde plus 1% osmium tetroxide in 0.1 M S-Collidine buffer, pH 7.3 (Kistler et al., 1970). They were postfixed for 20 rain in 0.25% uranyl acetate in 0.025 M veronal acetate buffer, pH 6,3, then detached with a rubber policeman, embedded in agar and dehydrated through an ethanol series. The cell/agar pellet was embedded in Epon, sections were cut at 40-50 nm and double-stained with uranyl acetate and lead citrate.

b) Scanning EM. The cells were transferred to petri dishes with a base of FEP-Tefion (Heraeus ,,Petriperm"), and allowed to reach confluency. They were fixed as described above and the bases supporting the cells were cut into 5 mm squares, which were processed through an ethanol series into an amylacetate intermedium. The specimens were critical-point-dried under CO 2 pressure, then mounted on object holders and their edges painted with conductive silver. They were coated with gold in a sputtering apparatus and examined in a Cambridge Stereoscan $4 scanning electron microscope.


R e s u l t s

Biochemistry

The activity of L D H (units per g protein) in all groups of cell cultures at the experimental stages examined is shown in Table 1. At each stage, enzyme levels were similar in negative control cells and in both groups of sham smoked cells, a l though activity was progressively reduced with time. In the two groups of smoke-exposed cells, L D H was increasingly inhibited with exposure. Activity in these cells, expressed as a percentage of the activity in the corresponding sham exposed cells at the same stage, is shown in Figure 1. Smoke f rom the Kentucky research cigarette produced a greater inhibition o f L D H (78% reduc- t ion in activity after 48 puffs) than that f rom the A S F C cigarette (64% reduct ion after 72 puffs).

Cytochemistry

L D H was localized as a granular fo rmazan deposit t h roughou t the cytoplasm of the cultured cells, a particularly intense reaction being observed in the region a round the nucleus (Fig. 2a). In all groups, the number o f enzyme-posit ive cells was clearly reduced with increasing periods in culture. A greater degree of reduct ion was evident in the smoke-exposed cultures, especially those exposed to Ken tucky cigarette smoke. The smoke-exposed cells which were L D H positive also demonst ra ted a much weaker reaction than control cells at the same stage

(Fig. 2b).

Electrophoresis

L D H in cells of all five groups of cultures migrated as four isozymes which had the same electrophoretic mobilities as rabbit serum L D H isozymes 2-5 (Fig. 3), and which were therefore numbered S IRC cell L D H 2 5. The sub-

Table 1. LDH activity in control and smoke-exposed SIRC ceils during the course of the experiment. Each value represents the arithmetic mean + S.E.M. of eight replicate samples from the pooled cell populations of two culture dishes

LDH activity in cultured SIRC cells (units per g protein)

Passage Negative Cultures exposed to smoke number control from ASFC cigarettes

cultures - not number sham exposed of exposed

puffs cells

Cultures exposed to smoke from the Kentucky cigarette

smoke- number sham smoke- exposed of exposed exposed cells puffs cells cells

5 3139 +- 54 33 3336 +_ 123 7 178l +_49 57 1691+_ 35 9 I190 +_ 34 72 1109 +_ 21

2320+60 22 3264+__111 2333+__56 646 +- 15 38 1405__+ 37 457+__10 394+_ 9 48 1325+ 99 281+ 5

Inhibition of Lactate Dehydrogenase by Cigarette Smoke

100

80-

Fig. l a and b. LDH activity in cells exposed to smoke from a) ASFC cigarettes or b) Kentucky cigarettes, expressed as a percentage of the activity in sham exposed controls at the same stage. Abscissa: total number of puffs. Ordinate: LDH activity as percentage of the activity in sham exposed controls, C = 100%

60-

40-

20-

C 33 57 72 C 22

311

38 48

Fig. 2a and b. Histochemical localization of LDH in cultured SIRC cells, a) Negative control, ninth passage, b) Exposed to 42 puffs of smoke from the Kentucky cigarette, ninth passage. • 1100


Fig. 3a-f . LDH isozymes in smoke-exposed and sham exposed SIRC cells (b-f) compared with the isozymes in rabbit serum (a). a) Rabbit serum, b) Negative control, ninth passage, e) Exposed to 72 puffs of air (sham exposed), d) Exposed to 72 puffs of smoke from ASFC cigarettes, e) Exposed to 48 puffs of air (sham exposed), t) Exposed to 48 puffs of smoke from Kentucky cigarettes

banding shown by serum isozyme 5 was never observed in LDH-5 of the cultured cells, but a clear zone in the g e l , - a "noth ing d e h y d r o g e n a s e " - w a s always present in the positio n of L D H - I . In control cells, the strongest staining reactiola was produced by isozyme 3, then 4, 5 and 2. A similar pattern was found for both groups of test cultures at the beginning of the experiment but this became modified with increasing exposure. Cells exposed to smoke from the ASFC Cigarette showed a gradual reduction in staining intensity of LDH-4 and 5 which were only just detectable after 72 puffs (Fig. 3d). In cells exposed t o Kentucky cigarette smoke, isozyme 2 appeared to be absent at the 48 puff stage, while 3 and 4 were observed as extremely faint bands (Fig. 3 f).

Morphology

Phase Contrast Microscopy. With exposure to smoke from each type of cigarette, the SIRC cells gradually lost their fibroblast-like morphology (Fig. 4a) and


Fig. 4a and b. SIRC cell cultures viewed in the phase contrast microscope, a) Negative controls, ninth passage. Note fibroblastic appearance of cells, b) Exposed to 42 puffs of Kentucky cigarette smoke, ninth passage. Note epitheloid appearance of the cells, x 390

assumed an epitheloid shape. This change was first noted in the sixth passage of the exposure programme (45 puffs ASFC; 30 puffs Kentucky), and by the end of the experiment, smoke-exposed cells had the typical appearance shown in Figure 4b. The cells were rounded and the large ovoid nuclei contained prominent nucleoli. No changes in morphology were observed in the negative controls or in sham exposed cell cultures.

Scanning Electron Microscopy. The surface features of smoke-exposed cells were similar to those of controls throughout the investigation. Zeiotic blebs were common and most of the cells displayed varying numbers of filopodia (Fig. 5).

Transmission Electron Microscopy. In general, the ultrastructure of control and test cells was consistent in all stages examined. No obvious qualitative or quanti- tative variations were observed in such structures as the Golgi complex, mono- and polyribosomes, multivesicular bodies and microfilaments (Fig. 6a, b and c). In all cells, the p lasmalemma regularly formed vacuole-like structures which were sometimes associated with small, membrane-bound vesicles, and which corresponded in size to the surface blebs observed in the scanning electron microscope (Fig. 5). The mitochondria of cells exposed to smoke from the Kentucky cigarette contained matrix granules which were both larger and more


Fig, 5. Scanning electron mlcrograph of a SIRC cell after exposure to 34 puffs of Kentucky cigarette smoke. FP Filopodia, M V Microvilli, Z B Zeiotic blebs. • 6400

Fig. 6a-c. Transmission electron micrographs of control and smoke-exposed SIRC ceils. Arrows indicate mitocho~driat matrix granules, a) After exposure to 34 puffs of Kentucky cigarette smoke. V Vesicles, Z B Zeiotic blebs, x 28,000. b) Negative control, same stage as in (a), M V B multivesicular body. x 30,000. e) After exposure to 50 puffs of Kentucky cigarette smoke. Note increased size and number of mitochondrial matrix granules. G Golgi complex, M V B multivesicular body. • 30,000.


numerous than those found in the mitochondria of cells exposed to the ASFC cigarette smoke, or in the controls (Fig. 6c). These matrix granules were found to accumulate with increasing exposure to smoke.

Discussion

During the course of the experiment, LDH activity in control SIRC cells became progressively reduced. This is thought to represent a metabolic adaptation of the cells to the specific culture conditions used. In cells exposed to smoke from the ASFC or the Kentucky research cigarettes, however, LD H became increasingly depressed when compared with the enzyme level in controls at the same stage. This smoke-induced inhibition of activity was consistent with the observed diminished cytochemical reaction for LD H and with the loss of specific isozymes. In addition to these biochemical changes, smoke-exposed cells gradually lost their fibroblastic appearance and became more epitheloid in shape. An accumulation of dense mitochondrial matrix granules was also observed in these cells, especially in those exposed to Kentucky cigarette smoke, which displayed the greater inhibition of LDH (Fig. 1).

The mechanism by which cigarette smoke exerts its effect on LD H is not known, but three main possibilities may be considered: 1) that smoke affects directly transcription or translation of the DNA sequences which code for the enzyme (two genes). This would be consistent with previous observations that cigarette smoke can modify DNA and RNA content and DNA synthesis (Leuchtenberger etal. , 1973a, 1973b, 1974; Holt etal . , 1975; Styles, 1976). In addition, it was noted in the present study that Kentucky cigarette smoke tended to suppress the fastest migrating LDH isozymes, whereas smoke from the ASFC cigarette inhibited the slowest ones. The functional molecule of each L DH isozyme consists of a different tetrameric combination of two subunits which are produced under the control of separate genes. Homologous combina- tions of each type of sub-unit represent the fastest and slowest migrating isozymes, although in SIRC cells the fastest (LDH-1) seems to be absent. The different effects of Kentucky and ASFC smoke on LD H isozymes could therefore represent a specific inhibition of the expression of a different gene by each type of smoke. 2) That smoke directly inactivates an already synthesized active form of LDH. 3) That smoke inhibits a reaction or reactions in a metabolic pathway which also includes the lactate/pyruvate reaction, so that LD H activity could be reduced by a feedback mechanism. The enlargement of the mitochondrial matrix granules observed in SIRC cells exposed to smoke from Kentucky cigarettes may reflect such an inhibition of enzyme activity. Studies on cation accumulation in mitochondria suggest that the granules are aggregates of diva- lent cations, most probably Ca 2+, or phosphate (Vasington and Murphy, 1962; Greenawalt et al., 1964). The presence of large amounts of Ca 2 § in the mitochondria is known to inhibit oxidative phosphorylation, a process which is coupled with the respiratory chain of the tricarboxylic acid cycle (De Robertis et al., 1975). A series of feedback inhibitory mechanisms may, therefore, be postulated, acting from the level of oxidative phosphorylation via the tricarboxylic acid to the lactate/pyruvate reaction, and resulting in a reduction in LDH activity.


The specific c o m p o n e n t or c o m p o n e n t s in c igaret te smoke respons ib le for the inh ib i t ion of L D H are unknown. Toxic and mutagen ic act ivi ty have been d e m o n s t r a t e d in bo th the par t i cu la te and gas -vapour phase of smoke (Inui and T a k a y a m a , 1971 ; Leuch tenberger et al., 1974; Benedict et al., 1975; Davies et al., 1975; Styles, 1976). In the present inves t igat ion, a greater inh ib i to ry effect was p r o d u c e d by smoke f rom the K e n t u c k y cigaret te than tha t f rom the A S F C cigaret te . This m a y reflect the greater " t a r " and nicot ine content o f the K e n t u c k y cigaret te , the higher number of b io logica l ly active c ompone n t s in its v a p o u r phase (Davies et al., 1975), the fact tha t its smoke was unfi l tered, or a c o m b i n a t i o n of some or all of these factors. We are current ly invest igat ing whether the cons t i tuents which modi fy L D H act ivi ty can be local ized in either the par t i cu la te or gas -vapour phase.

In an earl ier inves t iga t ion (F leming and Kist ler , 1977), we were unab le to detect morpho log i ca l changes or h i s tochemica l and i sozyme var ia t ions in several enzymes, inc luding L D H , in the Veto cell l ine exposed to smoke f rom the A S F C cigarette. In the present s tudy, smoke f rom the same cigaret tes and under the same exper imenta l condi t ions , p r o d u c e d a c lear ly d e m o n s t r a t e d inh ib i t ion of L D H in S I R C cells. This d iscrepancy in our f indings suggests tha t the results ob ta ined f rom in vi t ro exper iments of this na ture may depend largely on the cell line chosen for invest igat ion.

Acknowledgements. We thank Prof. Dr. H.R. Hohl, Institute of Plant Biology, University of Ziirich for allowing us to use his Cambridge S-4 Scanning electron microscope. The exc4Jlent technical assistance of Miss M. Balzer, Mr. W:F. Scherle and Miss E. Meier is gratefully acknowledged.

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Received November 25, 1977

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Inhibition of lactate dehydrogenase in cultured SIRC cells by cigarette smoke