Enhancement of oxidative damage to cultured cells and Caenorhabditis elegans by mitochondrial electron transport inhibitors.

The mechanisms that lead to mitochondrial damage under oxidative stress conditions were examined in primary and cultured cells as well as in the nematode Caenorhabditis elegans (C. elegans) treated simultaneously with electron transport inhibitors and oxygen gas. Oxygen loading enhanced the damage of PC 12 cells by thenoyltrifluoroacetone (TTFA, a complex II inhibitor), but did not by rotenone (a complex I inhibitor), antimycin (a complex III inhibitor), and sodium azide (a complex IV inhibitor). In primary hepatocytes, the enhancement was observed with the addition of sodium azide and rotenone, but not by TTFA or antimycin. In the nematode, only rotenone and TTFA enhanced the sensitivity under hyperoxia. These results demonstrate that highly specific inhibitors of electron transport can induce oxygen hypersensitivity in cell levels such as PC 12 cells and primary hepatocytes, and animal level of C. elegans. In addition the cell damage is different dependent on cell type and organism.

A defect in the cytochrome b large subunit in complex II causes both superoxide anion overproduction and abnormal energy metabolism in Caenorhabditis elegans.

A mev-1(kn1) mutant of the nematode Caenorhabditis elegans is defective in the cytochrome b large subunit (Cyt-1/ceSDHC) in complex II of the mitochondrial electron transport chain. We have previously shown that a mutation in mev-1 causes shortened life span and rapid accumulation of aging markers such as fluorescent materials and protein carbonyls in an oxygen-dependent fashion. However, it remains unclear as to whether this hypersensitivity is caused by direct toxicity of the exogenous oxygen or by the damage of endogenous reactive oxygen species derived from mitochondria. Here we report important biochemical changes in mev-1 animals that serve to explain their abnormalities under normoxic conditions: (i) an overproduction of superoxide anion from mitochondria; and (ii) a reciprocal reduction in glutathione content even under atmospheric oxygen. In addition, unlike wild type, the levels of superoxide anion production from mev-1 mitochondria were significantly elevated under hyperoxia. Under normal circumstances, it is well known that superoxide anion is produced at complexes I and III in the electron transport system. Our data suggest that the mev-1(kn1) mutation increases superoxide anion production at complex II itself rather than at complexes I and III. The mev-1 mutant also had a lactate level 2-fold higher than wild type, indicative of lactic acidosis, a hallmark of human mitochondrial diseases. These data indicate that Cyt-1/ceSDHC plays an important role not only in energy metabolism but also in superoxide anion production that is critically involved in sensitivity to atmospheric oxygen.

Mitochondrial mutations differentially affect aging, mutability and anesthetic sensitivity in Caenorhabditis elegans.

In the nematode Caenorhabditis elegans, mutations have been previously isolated that affect the activities of Complex I (gas-1) and Complex II (mev-1), two of the five membrane-bound complexes that control electron flow in mitochondrial respiration. We compared the effects of gas-1 and mev-1 mutations on different traits influenced by mitochondrial function. Mutations in Complex I and II both increased sensitivity to free radicals as measured during development and in aging animals. However, gas-1 and mev-1 mutations differentially affected mutability and anesthetic sensitivity. Specifically, gas-1 was anesthetic hypersensitive but not hypermutable while mev-1 was hypermutable but displayed normal responses to anesthetics. These results indicate that Complexes I and II may differ in their effects on behavior and development, and are consistent with the wide variation in phenotypes that result from mitochondrial changes in other organisms.

A comparison of mutations induced by accelerated iron particles versus those induced by low earth orbit space radiation in the FEM-3 gene of Caenorhabditis elegans.

The fem-3 gene of Caenorhabditis elegans was employed to determine the mutation frequency as well as the nature of mutations induced by low earth orbit space radiation ambient to Space Shuttle flight STS-76. Recovered mutations were compared to those induced by accelerated iron ions generated by the AGS synchrotron accelerator at Brookhaven National Laboratory. For logistical reasons, dauer larvae were prepared at TCU, transported to either Kennedy Space Center or Brookhaven National Laboratory, flown in space or irradiated, returned to TCU and screened for mutants. A total of 25 fem-3 mutants were recovered after the shuttle flight and yielded a mutation frequency of 2.1x10(-5), roughly 3.3-fold higher than the spontaneous rate of 6.3x10(-6). Four of the mutations were homozygous inviable, suggesting that they were large deletions encompassing fem-3 as well as neighboring, essential genes. Southern blot analyses revealed that one of the 25 contained a polymorphism in fem-3, further evidence that space radiation can induce deletions. While no polymorphisms were detected among the iron ion-induced mutations, three of the 15 mutants were homozygous inviable, which is in keeping with previous observations that high LET iron particles generate deficiencies. These data provide evidence, albeit indirect, that an important mutagenic component of ambient space radiation is high LET charged particles such as iron ions.

Oxidative stress pretreatment increases the X-radiation resistance of the nematode Caenorhabditis elegans.

Pre-exposure of wild-type Caenorhabditis elegans to oxygen conferred a protective effect against the lethality imposed by subsequent X-irradiation. In contrast, two mutants (rad-1 and rad-2) that are UV and ionizing radiation hypersensitive but not oxygen sensitive, did not exhibit this adaptive response. To explore the molecular basis of protection, the expression of several key genes was examined using Northern blot analyses to measure mRNA levels. In the wild-type, expression of the heat shock protein genes, hsp16-1 and hsp16-48, increased dramatically after incubation under high oxygen. Expression of two superoxide dismutase genes (sod-1 and sod-3) was relatively unaffected. Unlike the wild-type, the basal levels of these four genes were significantly lower in the rad-1 and rad-2 mutants under atmospheric conditions. These genes were partially induced in response to oxidative stress. These data suggest that at least a portion of the hypersensitive phenotype of rad-1 and rad-2 may be attributed to inappropriate gene expression.

Lethal consequences of simulated solar radiation on the nematode Caenorhabditis elegans in the presence and absence of photosensitizers.

The partial destruction of the earth's protective ozone layer has raised concerns about the impact of increased UV radiation on the earth's biological systems. In this study, polychromatic light sources were employed to observe the biological responses of the soil nematode Caenorhabditis elegans to simulated solar UV. Using various filter combinations, action spectra were constructed that approximated those generated previously with mono-chromatic radiation. In both cases, a mutant strain (rad-3) progressively lost its hypersensitivity as shorter wave-lengths were filtered out. In addition, both wild type and radiation-sensitive (rad) mutants were irradiated with several combinations of filtered light sources in the presence and absence of two exogenous photosensitizers (ethidium bromide and bromodeoxyuridine). Treatment with either of the introduced photosensitizers increased photosensitivity to solar UV. Solar UV also induced a fluence-dependent reduction in fertility in wild-type animals. These experiments extend previous data and substantially expand our understanding of the biological responses of C. elegans to solar radiation.

A mutation in succinate dehydrogenase cytochrome b causes oxidative stress and ageing in nematodes.

Much attention has focused on the aetiology of oxidative damage in cellular and organismal ageing. Especially toxic are the reactive oxygen byproducts of respiration and other biological processes. A mev-1(kn1) mutant of Caenorhabditis elegans has been found to be hypersensitive to raised oxygen concentrations. Unlike the wild type, its lifespan decreases dramatically as oxygen concentrations are increased from 1 to 60%. Strains bearing this mutation accumulate markers of ageing (such as fluorescent materials and protein carbonyls) faster than the wild type. We show here that mev-1 encodes a subunit of the enzyme succinate dehydrogenase cytochrome b, which is a component of complex II of the mitochondrial electron transport chain. We found that the ability of complex II to catalyse electron transport from succinate to ubiquinone is compromised in mev-1 animals. This may cause an indirect increase in superoxide levels, which in turn leads to oxygen hypersensitivity and premature ageing. Our results indicate that mev-1 governs the rate of ageing by modulating the cellular response to oxidative stress.

Replication in UV-irradiated Caenorhabditis elegans embryos.

Replication continues in wild-type (but not rad mutant) Caenorhabditis elegans embryos even after exposure to massive fluences of UV radiation. It is of interest to elucidate the mechanism(s) for this "damage-resistant" DNA synthesis. In this study, DNA from unirradiated and UV-irradiated wild-type embryos was examined using the electron microscope. Large fluences of UV radiation (180 J m-2) had little effect on either replication bubble size or distances between bubbles in wild-type embryos, indicating that the damage-resistant DNA synthesis was not grossly aberrant. Conversely, UV irradiation significantly decreased center-to-center distances between bubbles in excision-repair-deficient rad-3 embryos. This suggests that the decreased DNA synthesis observed after UV irradiation in rad-3 embryos is due largely to blockage of elongation of DNA synthesis.

Genetic and molecular analyses of UV radiation-induced mutations in the fem-3 gene of Caenorhabditis elegans.

The utility of a new target gene (fem-3) is described for investigating the molecular nature of mutagenesis in the nematode Caenorhabditis elegans. As a principal attribute, this system allows for the selection, maintenance and molecular analysis of any type of mutation that disrupts the gene, including deletions. In this study, 86 mutant strains were isolated, of which 79 proved to have mutations in fem-3. Twenty of these originally tested as homozygous inviable. Homozygous inviability was expected, as Stewart and coworkers had previously observed that, unlike in other organisms, most UV radiation-induced mutations in C. elegans are chromosomal rearrangements of deficiencies (Mutat. Res. 249, 37-54, 1991). However, additional data, including Southern blot analyses on 48 of the strains, indicated that most of the UV radiation-induced fem-3 mutations were not deficiencies, as originally inferred from their homozygous inviability. Instead, the lethals were most likely "coincident mutations" in linked, essential genes that were concomitantly induced. As such, they were lost owing to genetic recombination during stock maintenance. As in mammalian cells, yeast and bacteria, the frequency of coincident mutations was much higher than would be predicted by chance.

The effects of temperature on the longevity of a radiation-sensitive mutant rad-8 of the nematode Caenorhabditis elegans.

The effects of temperature on development and life span were examined in a radiation- and oxygen-hypersensitive mutant (rad-8) of the nematode Caenorhabditis elegans. At temperatures greater than 20 degrees C, the rad-8 mutant developed slightly slower and possessed a life span roughly equivalent to that of the wild type. At 16 degrees C, however, the mutant lived considerably longer than the wild type, with mean life spans of approximately 28 and 21 days, respectively. This lengthened life span was due to slower development. It was also dependent upon oxygen concentration, because the mean life spans of rad-8 and wild type were experimentally identical when reared at 16 degrees C in the presence of 5% rather than atmospheric oxygen. The rad-8 mutant represents an interesting paradox, as its life span can either be shortened or lengthened relative to the wild type, depending on temperature and oxygen concentration.

The radiation-sensitive mutant rad-8 of Caenorhabditis elegans is hypersensitive to the effects of oxygen on aging and development.

A mutant of rad-8, originally isolated on the basis of its hypersensitivity to ultraviolet radiation, is also hypersensitive to oxygen and methyl viologen, a superoxide-anion generator. Oxygen also retarded development and reduced fecundity in a concentration-dependent fashion in rad-8 but not in wild type. In addition, the mean life span of rad-8 (but not wild type) was progressively shortened when animals incubated in increasing oxygen concentrations. This cross hypersensitivity to both UV radiation and free radicals provides further evidence that DNA damage may be important in the aging process.

Inactivation of wild-type and rad mutant Caenorhabditis elegans by 8-methoxypsoralen and near ultraviolet radiation.

Survival of wild-type and four radiation-sensitive (rad) mutants of the nematode Caenorhabditis elegans was determined after near-UV irradiation in the presence of 8-methoxypsoralen (8-MOP). Three sets of inactivation profiles were generated for each strain by irradiating synchronous populations of either early embryos, late embryos or first-stage larvae (L1s). Late embryos were consistently the most sensitive. Curiously, none of the four rad mutants were even moderately hypersensitive. Split-dose experiments indicated that DNA-DNA crosslinks were primarily responsible for lethality. Crosslink induction and repair were determined using two different assays. In both cases, little if any repair was observed in wild-type. This lack of repair thus explains why the rad mutants were not hypersensitive to 8-MOP photoinactivation. Since early embryos undergo extensive cell cycling, their resistance to 8-MOP photoinactivation suggests that replication is highly refractory to both monoadducts and crosslinks, as has been demonstrated previously for UV radiation-induced photoproducts (Hartman et al., 1991, Mutat. Res., 255, pp. 163-173).

The regulation of DNA repair during development.

DNA repair is important in such phenomena as carcinogenesis and aging. While much is known about DNA repair in single-cell systems such as bacteria, yeast, and cultured mammalian cells, it is necessary to examine DNA repair in a developmental context in order to completely understand its processes in complex metazoa such as man. We present data to support the notion that proliferating cells from organ systems, tumors, and embryos have a greater DNA repair capacity than terminally differentiated, nonproliferating cells. Differential expression of repair genes and accessibility of chromatin to repair enzymes are considered as determinants in the developmental regulation of DNA repair.

Excision repair of UV radiation-induced DNA damage in Caenorhabditis elegans.

Radioimmunoassays were used to monitor the removal of antibody-binding sites associated with the two major UV radiation-induced DNA photoproducts [cyclobutane dimers and (6-4) photoproducts]. Unlike with cultured human cells, where (6-4) photoproducts are removed more rapidly than cyclobutane dimers, the kinetics of repair were similar for both lesions. Repair capacity in wild type diminished throughout development. The radioimmunoassays were also employed to confirm the absence of photoreactivation in C. elegans. In addition, three radiation-sensitive mutants (rad-1, rad-2, rad-7) displayed normal repair capacities. An excision defect was much more pronounced in larvae than embryos in the fourth mutant tested (rad-3). This correlates with the hypersensitivity pattern of this mutant and suggests that DNA repair may be developmentally regulated in C. elegans. The mechanism of DNA repair in C. elegans as well as the relationship between the repair of specific photoproducts and UV radiation sensitivity during development are discussed.

Radiation effects on life span in Caenorhabditis elegans.

Wild-type and radiation-sensitive (Rad) mutants of Caenorhabditis elegans were irradiated using a 137Cs source (2.7 krads/min.) at several developmental stages and subsequently monitored for life span. Acute doses of radiation ranged from 1 krad to 300 krads. All stages required doses above 100 krads to reduce mean life span. Dauers and third stage larvae were more sensitive, and 8-day-old adults were the most resistant. Occasional statistically significant but nonrepeatable increases in survival were observed after intermediate levels of irradiation (10-30 krads). Unirradiated rad-4 and rad-7 had life spans similar to wild-type; all others had a significant reduction in survival. The mutants were about as sensitive as wild-type to the effects of ionizing radiation including occasional moderate life span extensions at intermediate doses. We conclude that the moderate life span extensions sometimes observed after irradiation are likely to be mediated by a means other than the induction of DNA repair enzymes.

An endonuclease from Caenorhabditis elegans: partial purification and characterization.

A deoxyribonuclease was partially purified from the free-living nematode Caenorhabditis elegans. The DNase functioned as an endonuclease and introduced both single-strand nicks and double-strand breaks into DNA. The enzyme hydrolyzed double-stranded DNA seven times more rapidly than single-stranded DNA. DNase activity was not affected by the addition of divalent cations below 1 mM but was inhibited at higher ionic concentrations. In addition, the enzyme was not inhibited in the presence of 10 mM EDTA. The enzyme was inhibited by salt concentrations greater than 20 mM. Three independent mutations in the nuc-1 gene were shown to reduce nuclease activity to less than 1% of that seen in wild-type organisms.

Radiation sensitivity and DNA repair in Caenorhabditis elegans strains with different mean life spans.

The sensitivities to three DNA damaging agents (UV and gamma-radiation, methyl methanesulfonate) were measured in four recombinant inbred (RI) strains of Caenorhabditis elegans with mean life spans ranging from 13 to 30.9 days, as well as in the wild-type strains used to derive these RI's. Sensitivities at several stages in the developmental cycle were tested. There were no significant correlations between mean life span and the lethal effects of these 3 agents. Excision of two UV-radiation-induced DNA photoproducts was also measured. Long-lived strains were no more repair competent than shorter-lived strains. These data indicate that DNA repair plays at best a minor role in the aging process of C. elegans.