Changes in the DNA synthesis pattern of paramecium with increased clonal age and interfission time.

The clonal age in paramecia refers to the total number of vegetative divisions a clone has undergone since its origin at autogamy (self-fertilization). As clonal age increases, the interfission time usually increases. The DNA synthesis pattern of cells of different ages was compared by autoradiographic analysis of the DNA synthesis of synchronized cells at various time intervals during the cell cycle (from one division to the next). The study showed that the G(1) period (the lag in DNA synthesis post division) was constant, irrespective of interfission time or clonal age; but the duration of the DNA synthesis period increased with increased interfission time or clonal age. Therefore, we have shown for the first time that the G(1) period is fixed, and the S period is increased in a eukaryotic unicellular organism as a function of interfission time and clonal age.

Loss of endocytic capacity in aging Paramecium. The importance of cytoplasmic organelles.

Aged cells have significantly fewer food vacuoles and ingest fewer bacteria than young cells. Loss of food vacuoles was explained by a decreasing difference in the food vacuole formation and excretion rates; the formation rate declined more rapidly than the excretion rate, approaching equivalence at 160 fissions, when the proportion of cells with no food vacuoles, in the presence of excess food, abruptly increased. A model for cellular aging is presented in which control of organelle numbers and cyclical interactions between the nucleus and cytoplasm may be of critical importance.

DNA repair and longevity assurance in Paramecium tetraurelia.

At given doses and clonal ages, ultraviolet irradiation-induced DNA damage reduced clonal life-span, but when followed by photoreactivation, extension of clonal life-span was observed. If photoreactivation preceded the ultraviolet treatment, no significant beneficial effect was detected. Because studies of others have shown that photoreactivation repair monomerizes the ultraviolet-induced cyclobutane dimers in DNA, but does not affect the other photoproducts, these results indicate that DNA damage can influence the duration of clonal life-span unless that damage is repaired. Repeated treatment with ultraviolet and photoreactivation resulted in significant mean and maximal clonal life-span extension when compared with untreated controls, and it is assumed that the rejuvenation effect was due to the correction or prevention of some age damage.

Mutagenicity of fly ash particles in Paramecium.

Paramecium, a protozoan that ingests nonnutritive particulate matter, was used to determine the mutagenicity of fly ash. Heat treatment inactivated mutagens that require metabolic conversion to their active form but did not destroy all mutagenicity. Extraction of particles with hydrochloric acid, but not dimethyl sulfoxide, removed detectable mutagenic activity.

Age-correlated changes in expression of micronuclear damage and repair in Paramecium tetraurelia.

In Paramecium, age is defined as the number of mitotic divisions which have elapsed since the previous cross-fertilization (conjugation) or self-fertilization (autogamy). As the mitotic interval between fertilizations increases, the percentage of nonviable progeny clones increases. In the current study, resolution of conflicting previous reports on the pattern of increase of death and reduced viability in progeny from aging parent cells is found. Some exautogamous clones exhibit a high mortality at young clonal ages, others show no mortality throughout their life span, but most (73%) show an abrupt increase in the percent death and reduced viability in progeny from cells 50-80 fissions old. Ultraviolet-irradiation-induced micronuclear mutations, repairable by photoreactivation, increased with increased clonal age when monitored by percent death and reduced viability of exautogamous progeny of irradiated cells. Loss of dark repair is considered a contributor to the increased expression of micronuclear mutations with increased clonal age.

Nucleolar changes in aging and autogamous Paramecium tetraurelia.

A significant loss in nucleolar volume density (proportion of nuclear volume occupied by nucleoli) occurs in the macronucleus as clonal age increases, which suggests an age-correlated loss of ribosomal RNA synthesizing capacity. In the macronuclear fragments of cells undergoing autogamy, however, a significant gain in nucleolar volume density takes place.

Bioassay of genotoxic effects of environmental particles in a feeding ciliate.

The ciliated protozoan, Paramecium, can be used to quantitate cytotoxic and genotoxic effects of ingested complex environmental particles. Cytotoxicity is quantitated by the increased proportion of treated versus control cells which do not retain their capacity for normal cell replication. Genotoxic effects are assessed by the increased fraction of nonviable offspring from treated versus control parent cells after the self-fertilization process of autogamy. Since these cells ingest nonnutrient respirable-sized particles, biological activity of intracellular extraction of dusts and fly ash can be compared before and after extraction with polar and nonpolar solvents. Previous studies indicated that coal fly ash was mutagenic in these eukaryotic cells. Mutagenicity of coal fly ash was not detectable after extraction with a concentration of HCl known to remove nonmatrix trace elements. These results suggested that this ciliate bioassay might be a detector of mineral mutagens. Fine particles of the carcinogenic nickel compounds, alpha-nickel subsulfide, and beta-nickel sulfide were compared for their biological activity in this bioassay. Both nickel compounds were ingested by the ciliates and induced heritable damage in the progeny of the treated parent cells.

Longevity in the protozoa.

In ciliates there are examples of cells which have different proliferation potential in the macronucleus. Those species with limited macronuclear proliferation potential require sex to activate the reserve nucleus. In terms of the capital investment theory, some ciliates invested in their spare nucleus without loss of their original potential, while others accumulated debts and needed the reserve account to maintain life. Other cells neglected maintenance of their reserve account and failed unless their venture capital account was not a self-sustaining venture. Sex provided access to the reserve account and had to occur before deterioration of the reserve account. The question is not when cellular immortality was lost, but rather when immortality was partitioned from a mortal segment. The separation provided the option both for senescence and evolution in multicellular organisms. In colonial flagellates, separation of cells with infinite and finite cell lifespan potential occurred in some species, while in others the separation did not involve loss of immortality. In colonial flagellates, sex did not become an obligate stage. The immortal cells are haploid and could not accumulate damage and live (in contrast with the diploids in the ciliated protozoans). The present theory predicts that differences between species or cells with infinite versus finite lifespan potential may reveal differences in the critical determinants of longevity. Senescence could arise as an accident, as well as a design of nuclear differentiation. Cells therefore may have a much greater reserve for totipotency than would be predicted if they were assumed to lose immortality simply by the act of differentiation.

Malic dehydrogenase locus of Paramecium tetraurelia.

A search was undertaken for naturally occurring genetic markers for use in clonal aging studies of Paramecium tetraurelia. Clonal age is defined as the number of cell divisions since the last sexual process. Autogamy (self-fertilization) is a sexual process which can occur in aging lines, resulting in homozygosity and initiation of the next generation. Such "illicit" autogamies must be detected and eliminated from the aged clone. With codominant alleles, heterozygous aging lines can be established which will express a phenotype distinguishable from that of either parental type and autogamy can then be monitored by the appearance of either segregant homozygous phenotype. However, very few codominant alleles are available in this species. Electrophoretic mobilities of malic dehydrogenase (MDH) were assayed in 11 stocks of Paramecium tetraurelia by polyacrylamide gel electrophoresis. Nine stocks showed a single-banded "stock 51" type, while stock 174 and stock 29 each exhibited unique mobility. Crosses between stock 51 and the deviant stocks revealed distinct three-banded patterns indicative of heterozygosity of the F1 generation. In the autogamous F2 generation, 1:1 segregation of the parental types were recovered. The pattern of inheritance is consistent with codominant alleles and Mendelian inheritance. These naturally occurring biochemical markers are stable with increasing clonal age and are therefore useful genetic markers for studies of cellular aging.

Supplemental melatonin increases clonal lifespan in the protozoan Paramecium tetraurelia.

The hypothesis that melatonin supplementation can increase the lifespan of a single-celled organism was tested by the administration of melatonin to the ciliated protozoan Paramecium tetraurelia. Melatonin supplementation in dim red light at a dose of 0.043 mM (10 mg/L) of nutrient media (bacterized Cerophyl) per day, followed by incubation for 23 hr in darkness, increased the mean clonal lifespan of Paramecium tetraurelia in days by percentages ranging from 20.8% (P < 0.01, two-tailed t-test) to 24.2% (P < 0.01, ANOVA) over controls. Maximum clonal lifespan in days was also increased in melatonin-supplemented cells, from 14.8% to 24.0% over controls. Mean clonal lifespan in fissions was not significantly greater in melatonin-supplemented cells, with values ranging from 6.0% to 15.5% over controls. Maximum clonal lifespan in fissions did not differ appreciably, with values ranging from 1.0% to 9.1% over controls, except in the case of cells selected for rapid division rates, in which melatonin-supplemented cells (393 fissions) lived 20.9% longer than controls (325 fissions) in terms of cumulative cell doublings during the clonal lifespan. The finding that melatonin supplementation increased clonal lifespan in Paramecium tetraurelia, an aerobic, single-celled organism, suggests that the mechanism of melatonin's longevity-promoting effects may be intracellular.

Calendar life-span versus fission life-span of Paramecium aurelia.

The hypothesis that paramecia use fissions, not days, to measure length of cell life-span was investigated. Parallel cell lines were grown at 27 C and at 24 C. The daily fission rate of the cells at 24 C was lower than at 27 C. If the cells count fissions, not days, the life-span in fissions should remain unchanged, whereas the cell life-span in days should increase in the lines with reduced daily fission rate. The results showed a significant increase in cell life-span in days when the cells were cultivated for 70-100% of their life cycle at 24 C. The life-span as measured by fissions, however, remained unchanged regardless of the time of the life cycle when cells were shifted to 24 C. The data indicate that, as a model system for cellular aging, paramecia are comparable to cells which use cell doublings to measure life-span.

Effects of an extremely low frequency electromagnetic field on the cell division rate and plasma membrane of Paramecium tetraurelia.

The eukaryotic protozoan, Paramecium, was examined as a model for effects of pulsated electromagnetic fields (PEMF) on cells. A 72-Hz PEMF similar to fields employed clinically increased cell division rates in Paramecium by 8.5%. Two calcium transport mutants of these organisms showed differential responses to the same field. Verapamil, a calcium channel blocker, abolished any effect of PEMFs on cell division rates. A fluorescent probe that is thought to sense changes in membrane potential also manifested an altered response in the PEMF-exposed cells whereas a fluorescent lipid bilayer fluidity probe produced evidence of decreased membrane fluidity in the exposed cells. An effect of PEMFs on ion transport mediated by either a direct or indirect effect on the cell membrane is suggested by these studies.

Bioassay of environmental nickel dusts in a particle feeding ciliate.

The ciliated protozoan Paramecium was used to quantitate cytotoxic and genotoxic effects of nickel particles. The biological response of these eukaryotic cells to pure nickel powder and iron-nickel powder was assayed and compared to the effect of the inorganic carcinogen nickel subsulfide. Cytotoxicity was determined by the percent survival of treated cells. Genotoxicity was indicated by significant increases in the fraction of nonviable offspring (presumed index of lethal mutations) found after self-fertilization (autogamy) in parents from the nickel-treated versus neutral control groups. The cells were exposed to the dusts and the biological effects determined. Only the nickel subsulfide consistently showed a significant increase in offspring lethality.

Mutagenicity of coal fly ash: a new bioassay for mutagenic potential in a particle feeding ciliate.

The use of the established mutagenesis assay in Paramecium as a prescreen for hazardous environmental particles is described. Since these protozoans ingest particles of the size respired by animals and man, the biological effects of the respirable fraction of fly ash particles were monitored in particle-feeding eukaryotic cells. Fly ash from coal combustion was utilized for these studies and was found to be mutagenic. The effects of physical and chemical treatment of the particle mutagenicity provided evidence for both heat-stable, heat-labile and acid extractable mutagenic agents.