An α-tocopherol dose response study in Paramecium tetraurelia

Mechanisms of Ageing and Development 125 (2004) 21–30

An ␣-tocopherol dose response study in Paramecium tetraurelia Peter J. Minogue, John N. Thomas∗ Biology Department, Northeastern Illinois University, 5500 North Saint Louis Avenue, Chicago, IL 60625, USA Received 13 April 2003; received in revised form 31 August 2003; accepted 7 October 2003

Abstract Vitamin E (d,l-␣-tocopherol) was administered to Paramecium tetraurelia in doses of 10, 100, 1000 and 10,000 mg/l throughout its clonal lifespan. ANOVA revealed significant differences in clonal lifespan between groups, whether lifespan was measured in total fissions, or in days (P < 0.05). When mean clonal lifespan was measured in fissions the greatest difference was between the 1000 mg/l ␣-tocopherol treatment at 382 fissions, and the ethanol control at 255.5 fissions. The greatest difference in mean clonal lifespan in days survived was between the 10,000 mg/l ␣-tocopherol treatment at 292.5 days and the ethanol control at 76 days. ANOVA also revealed significant differences (P < 0.05) in the initial cell fission rates between groups. At the 1000 and 10,000 mg/l concentrations of ␣-tocopherol, a decrease in cell fission rates was apparent early in the lifespan, but these rates began to increase gradually during the late clonal lifespan. Although no clonal toxicity effects were found in terms of decreasing life-expectancy, the 1000 and 10,000 mg/l treatment groups exhibited higher background mortality rates throughout their respective lifespans than did the control groups, which could represent a cytotoxic effect. © 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: ␣-Tocopherol; Vitamin E; Clonal lifespan; Paramecium tetraurelia; Dose–response

1. Introduction Harman is credited with being the first to propose that free radicals may be the primary causative agent of aging (Harman, 1956; Arking, 1998). Subsequent research using free radical-quenching antioxidants in various animal models has provided support for this theory (Finch, 1990; Arking, 1998). The antioxidant Vitamin E in particular was shown to increase the lifespan in numerous organisms, including the nematode Caenorhabditis elegans (Epstein et al., 1972; Zuckerman and Geist, 1983; Harrington and Harley, 1988), the rotifers Asplanchna brightwelli (Sawada and Enesco, 1984; Sawada and Carlson, 1987) and Philodina (Enesco and Verdone-Smith, 1980), the fruit fly Drosophila melanogaster (Miquel et al., 1973), the banana fruit fly Zaprionus paravittiger (Kakkar et al., 1996), the rat Rattus norvegicus (Porta et al., 1980), the C3H/He and LAF1 inbred mice (Blackett and Hall, 1981), and the protist Paramecium tetraurelia (Thomas and Nyberg, 1988). A few studies in mice, however, showed no significant effect of Vitamin E on lifespan (Ledvina and Hodanova, 1980; Blackett and Hall, ∗ Corresponding author. Tel.: +1-773-442-5744; fax: +1-773-442-5730. E-mail address: [email protected] (J.N. Thomas).

1980; Lipman et al., 1998). Most studies on Vitamin E have only examined a narrow dose range and provide limited information on the safety, efficacy, and possible toxicity of this vitamin (Kappus and Diplock, 1992). Additionally, several studies that have found toxic effects used the ␣-tocopherol acetate form of Vitamin E (Kappus and Diplock, 1992), which is known to occur naturally only in the squash beetle Epilachna borealis (Attygalle et al., 1996). When the more commonly occurring form of Vitamin E (␣-tocopherol) has been examined, no clear indication of toxicity has been apparent (Kappus and Diplock, 1992). A previous study of ␣-tocopherol by Thomas and Nyberg (1988), found that supplementation could increase the lifespan of P. tetraurelia, but doses greater than 1000 mg/l were not tested. By performing a dose response curve examining concentrations of ␣-tocopherol both above and below this level, the present study has attempted to address three issues: could very high doses of ␣-tocopherol increase Paramecium lifespan even further than previous experiments, could high concentrations of the vitamin result in toxic effects, and is there an optimal dose of the vitamin for lengthening clonal lifespan in Paramecium. The ␣-tocopherol form of Vitamin E was selected since it has the highest biological activity (Traber and Arai, 1999). In addition, ␣-tocopherol and other forms of Vitamin E are

0047-6374/$ – see front matter © 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mad.2003.10.008

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lipid soluble and are localized in various cellular membranes (Wang and Quinn, 1999). The inner mitochondrial membrane in particular is an important site for Vitamin E localization, since this organelle generates large amounts of free radicals during electron transport and oxidative phosphorylation (Lenaz, 1998). Due to the high concentration of polyunsaturated fatty acids in the inner mitochondrial membrane, self-propagating lipid peroxidation reactions may be common (Finkel, 1997). Since ␣-tocopherol appears to be the most effective form of Vitamin E at preventing these lipid peroxidation reactions (Traber and Packer, 1995; Liebler and Burr, 2000), it was selected for dose response studies throughout the clonal lifespan of P. tetraurelia. Normally, Paramecium exhibits a non-senescent aging pattern with a constant probability of death throughout the lifespan (Comfort, 1979). Utilizing Sonneborn’s methodology, a senescent or type I clonal aging pattern was induced in P. tetraurelia (Thomas and Nyberg, 1988). The senescent pattern can be maintained by sustaining high nutrient levels through daily feeding of the Paramecium, which prevents the advent of autogamy (Sonneborn, 1954). The resulting clonal senescence was tracked for the various treatment groups by recording daily fission rates, mortality rates, cumulative fissions, and total days survived (calendar age). These measurements provided several comparisons among the various treatment groups, allowing for detailed study of the effects of different doses of ␣-tocopherol at the cellular level in P. tetraurelia.

2. Materials and methods 2.1. Solutions preparation Cerophyl medium was prepared by boiling 5.0 g of Cerophyl (Agri-Tech Inc., Kansas City, MO) and 1.5 g Na2 HPO4 in 2 l of deionized water. This mixture was allowed to boil for 5 min. Subsequently, the solution was filtered twice through a Buchner funnel with Whatman #1 qualitative filter paper. The ␣-tocopherol stock solution was prepared by dissolving a 5.0 g ampule of liquid (d,l)-␣-tocopherol (Sigma) with 5 ml of 100% ethanol for a total volume of 10 ml. This 500.0 mg/ml parent solution was placed in a plastic centrifuge vial and thoroughly wrapped in aluminum foil to reduce photo-oxidation of the vitamin. Prior to cell transfer, the appropriate volume of this stock solution was added to the bacterized Cerophyl media and vortexed. Four experimental groups were used at 10, 100, 1000 and 10,000 mg/l of ␣-tocopherol. A final 1% ethanol concentration was used to keep the ␣-tocopherol in solution. The two controls (non-ethanol and ethanol controls) were used in this experiment to determine if the ␣-tocopherol solubilizing agent, ethanol, was having any effect on Paramecium longevity (Table 1). Cerophyl media was inoculated with Kiebsiella pneumoniae and incubated at 28 ◦ C for 24 h prior to feeding the

Paramecium. After the 24 h growth period, the bacterized Cerophyl was distributed between six centrifuge vials. Each vial was then labeled, and the appropriate amounts of ethanol and/or ␣-tocopherol were added. Newly bacterized Cerophyl was used to prepare these solutions each day of the experiment. 2.2. Experimental procedure Stocks of P. tetraurelia of opposite mating types were obtained from the American Type Culture Collection (ATCC numbers 30567 and 30568). The cells were stimulated to conjugate by not feeding them any additional Cerophyl inoculated with K. pneumoniae for 48 h prior to mixing of opposite mating types (Sonneborn, 1954). Establishment of conjugating pairs is commonly used as a standard method of determining the start of a new life cycle in Paramecium (Nanney, 1974; Sonneborn, 1974; Smith-Sonneborn, 1981, 1984). Two conjugating pairs were selected and allowed to divide until each ex-conjugant had produced a minimum of 96 cells. This generated two separate replicate lines that were distributed in groups of 16 between the six treatment groups (Table 1). For example, the non-ethanol control consisted of 16 cells which had been selected randomly from among 96 of the 128 cells in replicate line I (the seventh generation descendants of one ex-conjugant from the first conjugating pair) and 16 cells which had been selected randomly from among 96 of the 128 cells in replicate line II (the seventh generation descendants of one ex-conjugant from the second conjugating pair). Each of the remaining treatment groups (the ethanol control, the 10, 100, 1000 and 10,000 mg/l groups) were established in the same manner. Since each cell was isolated into its own well of Cerophyl on a depression slide, the total number of cells appearing in that well on the following day provided a measure of cell division rates and cell mortality rates, as well as a potential source of cells for perpetuating that particular replicate line in that particular treatment group (Thomas and Nyberg, 1988). On a daily basis, the cells were removed from the 28 ◦ C incubator and counted under a stereomicroscope (Cambridge Instruments). Mortality data and fission rates were recorded for each treatment group. The cells with normal morphology Table 1 The replicate mean survivorship in days and fissions for ␣-tocopherol supplemented and unsupplemented groups Treatment group

Mean clonal lifespan in fissions ± standard error

Control Ethanol control 10 mg/l 100 mg/l 1000 mg/l 10,000 mg/l

278.5 255.5 238 327.5 382 339

± ± ± ± ± ±

11 2.5 8 40.5 4 13

Mean clonal lifespan in days ± standard error 80 76 68.5 106 191 292.5

± ± ± ± ± ±

3 1 3.5 17 16 8.5

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and the highest daily fission rates within each treatment group of each replicate line were selected and transferred into newly bacterized Cerophyl media each day to perpetuate each replicate line in each treatment group as long as possible. For example, if one of the 16 wells in the ethanol control group (replicate line I) contained 32 cells, while the remaining fifteen wells contained 16 cells each, the well with 32 cells was selected to provide 16 cells in establishing 16 wells for the ethanol control group (replicate line I). An alternative example, not uncommon towards the end of the clonal lifespan, might include eight wells containing only two cells each, and the eight remaining wells containing fewer than two cells each. In this case, the 8 wells with 2 cells each would be selected to provide 16 cells in establishing 16 wells for the following day for that particular treatment group and replicate line. If fewer than 16 cells were available, then fewer than 16 wells could be established for that treatment group and replicate line. Normal morphology consisted of cells with oval shapes and normal motility (Takagi and Yoshida, 1980). After the daily counts and cell transfers, the cells were placed back into the 28 ◦ C incubator for another night. These procedures were repeated until the last remaining cells in each treatment group of each replicate line had died, defining its clonal lifespan (Smith-Sonneborn and Reed, 1976; Smith-Sonneborn, 1981). 2.3. Data collection and graphing techniques The mortality data were collected by counting the number of cells that had failed to survive from the previous day. For example, if two of the 16 wells in the ethanol control group of replicate line II had zero cells after 24 h, and the remaining fourteen wells had 1 or more cells, then the 24 h mortality rate for the ethanol control group of replicate line II would be calculated as 2/16, or 12.5%, for that particular day. The fission rate data were collected by counting the number of paramecium cells descended from each cell that had been transferred 24 h earlier. For example, if one of the 16 wells in the ethanol control group of replicate line II had 32 cells after 24 h, and the remaining fifteen wells had less than 32 cells each, then the 24-h fission rate for the ethanol control group of replicate line II would be calculated as the logarithm base 2 of 32, or 5 fissions, for that particular day. The fission results were recorded for each of the wells, and whichever wells contained the most cells within a particular treatment group and replicate line were selected for fission rate analysis. Sometimes, especially towards the end of the clonal lifespan, more than one well would be required to transfer cells for the next day’s set of 16 wells, and if the number of cells in each well differed, they were both recorded for fission rate analysis (daily fission rates would then be averaged among all wells contributing to the next day’s transfers). In these cases, cells which had the highest cumulative fission rates were selected for transfers, rather than relying solely on the previous 24 h fission rates for cell selection. Survivorship data were calculated by using the

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mortality data, assuming an initial 100% survivorship, and computing the weekly percentage mortality rate. For example, if eight out of 16 wells had zero cells every day for a week in replicate I of the 10,000 mg/l ␣-tocopherol treatment group, then the mortality rate of 8/16, or 50% per day, could be used to calculate weekly survivorship by first determining daily survivorship (1–0.5), then multiplying this value by all other daily survivorship values for that week (raising 0.5 to the exponent of 7 in this case), giving a calculated weekly survivorship value of 0.0078125% for replicate line I of the 10,000 mg/l group in this particular case. All mortality, fission, and survivorship data were recorded on a daily basis and averaged over a weekly time interval. The weekly time interval was selected because daily values tend to oscillate slightly and this time interval is commonly used in the literature to smooth out such oscillations (Smith-Sonneborn and Reed, 1976; Thomas and Nyberg, 1988; Thomas and Smith-Sonneborn, 1997). Survivorship patterns that appeared to be different from the typical type I aging pattern were plotted on a semi-logarithmic scale in order to discern the aging pattern. 2.4. Statistical methods Maximum clonal lifespans in each replicate line of each treatment group were calculated both in terms of calendar age and cumulative fissions. In addition, the weekly fission rates were determined for all the treatment groups throughout their lifespan. The calendar age, cumulative fissions, and initial fission rate values were analyzed using single-factor ANOVA for treatments; a P-value of less than 0.05 was considered statistically significant (Devore, 1982). A two-tailed Student’s t-test was used to analyze the clonal lifespans in cumulative fissions and calendar age between the ethanol control and the other treatments; a P-value of less than 0.05 was considered statistically significant (Devore, 1982).

3. Results 3.1. Clonal lifespan data Single-factor ANOVA indicated statistically significant differences in clonal lifespans between treatment groups when measured in both days and fissions (P < 0.05). Student’s t-test analysis found that only the 1000 and 10,000 mg/l ␣-tocopherol treatments were significant when compared to the controls (P < 0.05). Table 1 summarizes the full experimental results of the mean clonal lifespan in days and fissions for each treatment group. Two controls were used in this experiment to see if ethanol was having any effect on Paramecium longevity. The non-ethanol control exhibited a replicate mean clonal lifespan of 278 ± 11 fissions and the ethanol control had a replicate mean clonal lifespan of 255.5 ± 2.5 fissions (Table 1). In terms of calendar age, the non-ethanol control

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between the ethanol control and the 1000 mg/l treatment. The replicate mean clonal lifespan for the control was 255.5±2.5, and 382 ±4 fissions for the 1000 mg/l treatment (Table 1), representing a 49.51% increase in the 1000 mg/l treatment over the control. 3.2. Survivorship

Fig. 1. A comparison of the mean replicate survivorship curves of all six of the treatment groups [non-ethanol control (䊐), ethanol control (䉱), 10 mg/l (), 100 mg/l (䊊), 1000 mg/l (䊏), and 10,000 mg/l (䊉) of ␣-tocopherol].

exhibited a replicate mean clonal lifespan of 80 ± 3 days, and the ethanol control had a replicate mean clonal lifespan of 76 ± 1 days (Table 1). Although differences between these two control groups were greatest when measured in fissions rather than days, neither measurement exhibited greater than a 10% difference in clonal lifespan between the controls. Furthermore, Student’s t-test analysis does not show a significant difference in mean clonal lifespan measured in either days or fissions (P > 0.05) between the two control groups. The greatest difference in days survived (when comparing controls to experimentals), was between the means of the ethanol control and the 10,000 mg/l ␣-tocopherol treatment. The ethanol control had a replicate mean clonal lifespan of 76 ± 1 days and the 10,000 mg/l treatment had a replicate mean clonal lifespan of 292.5 ± 8.5 days (Table 1), almost a fourfold increase of the 10,000 mg/l over the ethanol control group. When mean clonal lifespan was measured in fissions (again when comparing controls to experimentals), the greatest difference was

The non-ethanol control, the ethanol control, and the 10 mg/l ␣-tocopherol treatment all exhibited similar calculated survivorship curves based on daily mortality rates (Fig. 1). They showed high survivorship until the late lifespan, when they shifted to a rapid increase in cell death. This rapid decline appears to occur at approximately 63 days among the three groups (Fig. 1). The 100 mg/l ␣-tocopherol treatment showed a similar calculated survivorship curve, except the clones survived to a greater clonal lifespan in days and fissions (Fig. 1). The 1000 mg/l ␣-tocopherol treatment exhibited a unique calculated survivorship curve when compared to the controls (Fig. 1). This group initially showed a rapid decrease in survivorship that appeared to level off for most of the lifespan, only gradually decreasing. This trend continued until the late lifespan when the survivorship showed a rapid decrease, similar to that exhibited by the ethanol control. The calculated survivorship curve for the 10,000 mg/l ␣-tocopherol treatment exhibited a dramatic decrease in survivorship in the early lifespan (Fig. 1). This decrease resulted in only about 10% survivorship by day 100. For the rest of the lifespan, there was a very gradual decrease in the survivorship, declining from 10% survivorship to clonal extinction over a period of about 200 days (Fig. 1). This treatment exhibited a type II survivorship pattern, with a linear decline when plotted on a semi-logarithmic scale (Fig. 2). This pattern extended into the final days before clonal extinction when they exhibited a dramatic decrease

Fig. 2. A semi-logarithmic plot of calculated survivorship over time, comparing the mean replicate ethanol control group (䉱) to the 10,000 mg/l (䊉) of ␣-tocopherol experimental group.

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Fig. 3. A comparison of the mean replicate mortality curves of all six of the treatment groups [non-ethanol control (䊐), ethanol control (䉱), 10 mg/l (), 100 mg/l (䊊), 1000 mg/l (䊏), and 10,000 mg/l (䊉) of ␣-tocopherol].

in survivorship characteristic of the type I aging pattern (Kirkwood, 1985).

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transferred into the ␣-tocopherol solution. After this time time period, both replicates from the 1000 mg/l treatment had fluctuating mortality rates, which continued until the late lifespan, when the clones showed a rapid increase in mortality rates. The 10,000 mg/l treatment exhibited similar death rates to the 1000 mg/l treatment (Fig. 3), with a high initial mortality rate lasting for the first 40 days after being placed in the ␣-tocopherol solution. Unlike the replicates in the 1000 mg/l treatment, however, the 10,000 mg/l treatment replicates had a high initial mortality rate that almost led to the death of the clones. Replicate I dropped down to three cells on the third day and replicate II dropped down to 1 cell on the fifth day. After the initial shock, the cells recovered, but continued to maintain a highly variable mortality rate throughout the clonal lifespan. Unlike all the other treatments, the 10,000 mg/l treatment group never had a weekly mortality rate of zero. Late in the lifespan, the 10,000 mg/l treatment group showed a rapid increase in mortality that was similar to that of the other treatment groups (Fig. 3).

3.3. Mortality rates 3.4. Fission rates The non-ethanol control, the 10 mg/l treatment, and the 100 mg/l treatment all exhibited similar mortality curves when compared with the ethanol control (Fig. 3), although the 100 mg/l treatment survived for a longer period of time than the other groups (Fig. 3). Mortality rates for these groups were relatively low until the late lifespan when they shifted to an accelerating increase in mortality rates (Fig. 3). This rapid increase in mortality rate, which increased with each successive week, was exhibited by all six experimental groups late in the lifespan (Fig. 3). The 1000 mg/l treatment had an initially high mortality (Fig. 3) that lasted for about the first 40 days after being

The controls had weekly fission rates that started out high and gradually decreased over time, usually dropping by a few fissions with each successive week (Fig. 4). Both the 1000 and 10,000 mg/l treatment groups had weekly fission rates that started out low and slightly increased with each successive week until the latter half of the lifespan. By the last weeks of the lifespan, however, the mean weekly fissions dropped as the clones began to go extinct (Fig. 4). ANOVA data indicated statistically significant differences between the six treatment groups when the initial weekly fission rates were compared (P < 0.05).

Fig. 4. A comparison of the mean replicate weekly fission rates of all six of the treatment groups [non-ethanol control (䊐), ethanol control (䉱), 10 mg/l (), 100 mg/l (䊊), 1000 mg/l (䊏), and 10,000 mg/l (䊉) of ␣-tocopherol].

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4. Discussion Few studies have looked at long-term treatment with Vitamin E or the effects of high doses on aging or toxicity (Kappus and Diplock, 1992). Previous studies in mammals have suggested possible toxic effects in the ␣-tocopherol acetate form of the vitamin, which appear to be mostly due to the decreased absorption of Vitamins D and K, creating deficiency syndromes associated with these lipid-soluble vitamins (March et al., 1973; Abdo et al., 1986). These studies found that supplementation with Vitamins D and K in addition to ␣-tocopherol acetate could restore a normal state to the research organism. Human supplementation studies of Vitamin E are controversial, since most have occurred over short time periods and usually in already unhealthy subjects (Kappus and Diplock, 1992). Additional problems in examining potential toxic effects of the vitamin arise in the literature where many of the papers do not define the experimental form of Vitamin E being studied (Kappus and Diplock, 1992). For those few cases using the ␣-tocopherol form of Vitamin E, no decreases in lifespan have been reported, although most cases have been short term and utilized the acetate form of Vitamin E (Kappus and Diplock, 1992). The present study is generally in agreement with and extends the findings of a previous study on ␣-tocopherol supplementation in P. tetraurelia (Thomas and Nyberg, 1988). In that previous study, the highest dose of ␣-tocopherol administered (1000 mg/l) was associated with the longest clonal lifespans observed, both in terms of cumulative fissions (330 fissions for the 1000 mg/l group compared to 237 fissions for the control group) and in terms of calendar age (141 days for the 1000 mg/l group compared to 66 days for the control group). Maximum clonal lifespans in the present study exceed those found in the previous study (Thomas and Nyberg, 1988), both in terms of cumulative fissions and calendar age, in all treatment groups with comparable ␣-tocopherol doses (including controls). One possible reason for this increase in maximum clonal lifespans might be a higher effective ␣-tocopherol concentration in the nutrient media of the present study due to daily addition of Vitamin E, compared to the less frequent addition of Vitamin E and prolonged storage of ␣-tocopherol-containing nutrient media (several weeks) before new batches were prepared in the previous study. This newer protocol could have allowed a more uniform dispersion of ␣-tocopherol micelles over time. A second possible reason could be the isolation procedure, involving a protective rinse of the micropipette (previously sterilized in boiling, deionized water) in bacterized Cerophyl before transfer of paramecia in the present study, compared to immediate transfer of paramecia in a micropipette which had been sterilized in boiling, deionized water without the Cerophyl rinse in the previous study. The newer protocol may have protected the paramecia somewhat from high temperatures and/or osmotic shock. In support of these proposed procedural differences having an effect on clonal

lifespan are the lower apparent background mortality rates in the present study when compared to higher background mortality rates (in both controls and similar ␣-tocopherol dosage groups) in the previous study. Very high initial mortality rates for the 1000 mg/l ␣-tocopherol group were also found in the previous study, in agreement with similar findings for the 1000 and 10,000 mg/l ␣-tocopherol groups in the present study. This indication of a possible cytotoxic effect of ␣-tocopherol at the highest doses tested (1000 and 10,000 mg/l), leading to a near-extinction of both replicate lines in the 10,000 mg/l ␣-tocopherol group before their recovery in the first week of the experiment, is an important new finding which was not so clearly demonstrated at the maximum ␣-tocopherol concentration tested in the previous study (Thomas and Nyberg, 1988). In contrast to the possible cytotoxic effects of ␣-tocopherol, as evidenced by the high background mortality rates at the highest doses (Fig. 3), the hypothesis that ␣-tocopherol would exhibit clonal toxicity at high doses in P. tetraurelia was not fully supported in the present study. The treatment groups receiving 10,000 mg/l of the vitamin survived longer than the controls in both fissions and days, although their background mortality rates were higher as well. The high background mortality rates, which appear to correlate with the vitamin dosage, increasing from 100 to 1000 and to 10,000 mg/l ␣-tocopherol treatment groups (Fig. 3), could be considered a cytotoxic effect of high vitamin dosage. On the other hand, if one considers the longevity of the clonal population as a whole, there is a significant increase in clonal lifespan, measured in either fissions or days, when comparing the 1000 mg/l or 10,000 mg/l treatment groups to the ethanol control group (Table 1). This increased life-expectancy is most evident when clonal lifespan is measured in days, with a clear progression to longer calendar age at the highest Vitamin E doses tested (Table 1). When life-expectancy is measured in fissions, however, the highest dose of Vitamin E tested (the 10,000 mg/l group) exhibited a shorter clonal lifespan than the second-highest dose tested (the 1000 mg/l group), although both life-expectancies were still longer than those of the controls. This sub-optimal life-expectancy for the highest Vitamin E dosage group, when measured in cumulative fissions, suggests that ␣-tocopherol could conceivably exhibit clonal lifespan toxicity at even higher doses, but this suggestion of possible clonal toxicity remains a speculation until higher doses can be tested. The late life mortality data was similar between all six groups, with each group exhibiting a hyperbolic aging pattern (Fig. 3). This aging pattern is characterized by accelerating increases in mortality, resulting in a more rapid cohort extinction than would be predicted by the Gompertz pattern of uniform increases in mortality (Finch, 1990). The possibility that this late lifespan aging pattern is truly exhibited by P. tetraurelia cannot be answered by the present experiment, which lacks the necessary number of replicates per treatment to analyze late lifespan mortality patterns.

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However, the present data does suggest that progressive, detrimental changes have occurred within each cell line, ultimately resulting in rapid senescence towards the end of the clonal lifespan. These progressive changes leading to increased mortality rates were originally hypothesized to be based on the random segregation of polyploid macronuclear DNA fragments containing essential genes during amitotic fissions of the macronucleus (Sonneborn, 1954, 1978). Such random, amitotic segregation would be expected to lead to imbalanced macronuclear gene copy numbers in each successive generation, increasing the risk that some essential genes might ultimately be lost from the macronuclear genome of daughter cells late in the clonal lifespan, with correspondingly lethal effects. Random, amitotic segregation of macronuclear gene copies could account for the extremely high mortality rates observed towards the end of the clonal lifespan in P. tetraurelia, as well as the progressive decline in fission rates with aging (Sonneborn, 1978), but this mechanism does not fully account for the progressive increase in mortality rates which begins in the early clonal lifespan (Thomas and Nyberg, 1988; Thomas and Smith-Sonneborn, 1997), when macronuclear gene copy numbers, though imbalanced, should still be adequate to sustain all homeostatic functions necessary for cell survival (Thomas, 1996). An alternative, or supplementary hypothesis which could be at work here is genetic instability resulting from the loss of essential gene copies due to unrepaired DNA damage (Strehler, 1986). Although originally proposed to account for the age-associated decline in ribosomal DNA copy numbers in post-mitotic tissues of humans, dogs, and other animals (Strehler, 1986), unrepaired DNA damage could potentially explain the progressive increase in mortality rates throughout the clonal lifespan of single-celled organisms like P. tetraurelia as well (Holmes and Holmes, 1986; Smith-Sonneborn, 1987). If this proposed mechanism is at work during senescence in both single-celled and multicellular organisms, then the age-associated increase in mortality rates in P. tetraurelia might be due in large part to macronuclear genetic damage sustained from free radicals, which is then passed on during successive cell divisions (Smith-Sonneborn, 1990; Thomas, 1996). Such macronuclear genetic damage has been suggested by the finding of increased single-strand gaps, apurinic/apyrimidinic lesions, and double-strand breaks in the macronucleus of aging P. tetraurelia (Holmes and Holmes, 1986). Morphological changes in the macronucleus of aging Paramecium aurelia have also been reported (Sundararaman and Cummings, 1976), suggesting reduced molecular synthesis and transport in these cells. Although macronuclear DNA content is regulated during cell division in P. tetraurelia (Berger and Schmidt, 1978), and remains fairly uniform during the early clonal lifespan (Takagi and Kanazawa, 1982), the average size of macronuclear DNA fragments is markedly decreased by the late clonal lifespan, despite an apparent lack of telomere shortening (Gilley and Blackburn, 1994), supporting the hypothesis that an accumulation of macronuclear

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DNA damage may be the primary mechanism of aging in these cells (Holmes and Holmes, 1986). Eventually this damage might be expected to manifest itself as the inability of aging paramecia to adequately maintain all homeostatic systems needed for cell survival, since the number of optimally-functioning essential gene copies may be substantially reduced as macronuclear DNA fragments continue breaking into smaller pieces (Gilley and Blackburn, 1994). A progressive, age-related increase in free radical-mediated damage to macronuclear DNA fragments in P. tetraurelia could account for the limited number of cell divisions (Smith-Sonneborn and Reed, 1976; Takagi and Yoshida, 1980; Aufderheide, 1984; Takagi et al., 1987), as well as the limited number of macronuclear fissions (Heifetz and Smith-Sonneborn, 1981; Aufderheide and Schneller, 1985; Aufderheide, 1986/1987; Smith-Sonneborn, 1985), based on a gradual but accelerating loss of essential macronuclear genes during aging. Both the original hypothesis of random macronuclear gene segregation (Sonneborn, 1954, 1978), and the more recent hypothesis of unrepaired DNA damage to multiple copies of essential genes (Strehler, 1986; Holmes and Holmes, 1986), may be applicable to the increasing mortality rates and declining fission rates which accompany aging in P. tetraurelia. The present experiment supports the hypothesis that oxidative damage to macronuclear DNA contributes to aging in P. tetraurelia, since ␣-tocopherol supplementation would be expected to reduce the rate of oxidative damage to DNA (Zhang and Omaye, 2001). However, the present experiment also suggests that there may be an optimal dose of ␣-tocopherol for clonal longevity in P. tetraurelia. In this experiment, the 1000 mg/l treatment had the highest mean total fissions (382 ± 4 fissions) and the second highest mean calendar lifespan (191 ± 16 days). Concentrations that were lower or higher appeared to be sub-optimal for clonal lifespan extension. The lower concentration, 100 mg/l, survived for a shorter period in days and total fissions (Table 1), although one of the replicates of the 100 mg/l treatment group appeared to have died prematurely, perhaps due to poor cell selection at some point in the clonal lifespan. In the higher dose at 10,000 mg/l, clonal lifespan in cumulative fissions was not as great, but calendar lifespan was increased compared to the 1000 mg/l treatment. With Paramecium, total fissions are considered to be a better method of measuring clonal aging than the total lifespan in days, since calendar age can be increased simply by decreasing the cell division rate (Takagi and Yoshida, 1980; Aufderheide, 1986/1987; Fukushima et al., 1990). For example, by lowering the incubation temperature of the cells, the lifespan in days can be increased without any effect on lifespan in fissions (Smith-Sonnebom and Reed, 1976). However, the use of calendar age to describe clonal lifespan in paramecia can sometimes provide data which would not be accessible to cumulative fission analysis alone (Takagi et al., 1987), as in the case of non-dividing cells of P. tetraurelia which survived significantly longer in days when exposed to

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␣-tocopherol than to control media at the end of their clonal lifespans (Thomas and Nyberg, 1988). In the present experiment, the reduced fission rates in the 10,000 mg/l treatment probably accounts for some of the 284.87% increase in days survived over the ethanol controls. Furthermore, it probably contributed to the approximately 100 day increase in mean clonal lifespan in the 10,000 mg/l treatment compared to the 1000 mg/l treatment. This approximate 100 day increase in lifespan might also have contributed to the reduced lifespan in fissions in the 10,000 mg/l treatment compared to the 1000 mg/l treatment. With a longer lifespan in days, the 10,000 mg/l treatment may have incurred more free radical exposure than the 1000 mg/l treatment, which could have affected subsequent lifespan in fissions to a greater extent than subsequent lifespan in days. The 1000 and 10,000 mg/l ␣-tocopherol treatments both showed fission rates that were significantly reduced in the early lifespan compared to the controls (Fig. 4), although cell division rates improved somewhat as the cells became more accustomed to these very high ␣-tocopherol concentrations. Since cell division rates were reduced, and mortality rates were increased (when comparing the 1000 and 10,000 mg/l treatment groups to controls in the early clonal lifespan), the possibility of cytotoxicity due to oxidation of high-dose ␣-tocopherol must be considered (Culbertson et al., 2001; Cornwell et al., 2002; Sun et al., 2002). During free radical-mediated oxidation of ␣-tocopherol, it can be converted into the pro-oxidants ␣-tocopherol semiquinone radical and ␣-tocopherol quinone (Liebler and Burr, 2000; Culbertson et al., 2001; Herrera and Barbas, 2001; Van Haaften et al., 2001; Ikemoto et al., 2002; Szeto and Benzie, 2002), but reported cytotoxic effects from these oxidized ␣-tocopherol molecules are not as severe as those reported for oxidized ␥-tocopherol molecules (Cornwell et al., 2002; Calviello et al., 2003). If pro-oxidant forms of ␣-tocopherol were responsible for increased mortality rates and decreased fission rates in the high-dose treatment groups, then some type of hormesis effect could be postulated to account for the late-life recovery of improved fission rates in these groups (Calabrese and Baldwin, 2001). Hormesis effects following low dose ultraviolet or gamma radiation exposure in P. tetraurelia have been proposed previously (Smith-Sonneborn, 1979; Planel et al., 1987), based on induction of DNA-repair enzymes following radiation-induced DNA damage, but the recovery of fission rates and decline of mortality rates in the high dose Vitamin E treatments of the present study were more modest than those reported for ultraviolet or gamma radiation treatments. As shown in Figs. 3 and 4, the high dose Vitamin E treatment groups never did improve their fission rates or mortality rates to the full extent of early lifespan control groups, arguing for a limited role of hormesis in explaining the modest recovery from high mortality rates and low fission rates in the 1000 and 10,000 mg/l ␣-tocopherol treatment groups. Another possible mechanism for this reduction in cell division rates could be through inhibition of protein kinase C

activity. Reduced protein kinase C activity in the presence of ␣-tocopherol has been documented in smooth muscle cells (Boscoboinik et al., 1991, 1992), fibroblasts (Azzi et al., 1993), and monocytes (Venugopal et al., 2002), as well as many tumor cell lines (Steiner et al., 1997; Gopalakrishna and Jaken, 2000). Several calcium-dependent protein kinases have also been identified in Paramecium cells (Klumpp et al., 1990; Son et al., 1993; Kim et al., 1998), so it is conceivable that ␣-tocopherol induced reduction in cell division rates in P. tetraurelia could be mediated through a protein kinase C mechanism. Alternative mechanisms to explain the reduced proliferation of paramecia cells at high concentrations of ␣-tocopherol include: specific nuclear binding sites for ␣-tocopherol (Patnaik and Nair, 1977), activation of ␣-tocopherol specific cytosolic proteins (Guarnieri et al., 1980), and slowing of ATP synthesis in mitochondria by abstraction of electrons from the electron transport system (Traber and Packer, 1995). Future studies may elucidate which of these mechanisms, if any, contributes to reduced cell proliferation in paramecia treated with high doses of ␣-tocopherol. The present study suggests that ␣-tocopherol does not have clonal toxicity effects in P. tetraurelia at the highest doses tested (1000 and 10,000 mg/l). High background mortality rates at high doses of ␣-tocopherol, however, should be investigated in more detail at the beginning of the life cycle in Paramecium, since this could represent a cytotoxic effect. Intermediate doses of ␣-tocopherol could also be tested (between 100 and 10,000 mg/l) in an attempt to determine whether Vitamin E exhibits diminishing improvements in life-expectancy at high doses, in a manner similar to that of dose-dependent Vitamin A effects in aging D. melanogaster (Massie et al., 1993).

Acknowledgements The authors would like to thank the Biology Department at Northeastern Illinois University for funding the present study, and two anonymous reviewers for valuable comments on an earlier draft of this manuscript.

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