Effects of early life exposure to ultraviolet C radiation on mitochondrial DNA content, transcription, ATP production, and oxygen consumption in developing Caenorhabditis elegans
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Mitochondrial DNA (mtDNA) is present in multiple copies per cell and undergoes dramatic amplification during development. The impacts of mtDNA damage incurred early in development are not well understood, especially in the case of types of mtDNA damage that are irreparable, such as ultraviolet C radiation (UVC)-induced photodimers.
We exposed first larval stage nematodes to UVC using a protocol that results in accumulated mtDNA damage but permits nuclear DNA (nDNA) repair. We then measured the transcriptional response, as well as oxygen consumption, ATP levels, and mtDNA copy number through adulthood.
Although the mtDNA damage persisted to the fourth larval stage, we observed only a relatively minor ~40% decrease in mtDNA copy number. Transcriptomic analysis suggested an inhibition of aerobic metabolism and developmental processes; mRNA levels for mtDNA-encoded genes were reduced ~50% at 3 hours post-treatment, but recovered and, in some cases, were upregulated at 24 and 48 hours post-exposure. The mtDNA polymerase γ was also induced ~8-fold at 48 hours post-exposure. Moreover, ATP levels and oxygen consumption were reduced in response to UVC exposure, with marked reductions of ~50% at the later larval stages.
These results support the hypothesis that early life exposure to mitochondrial genotoxicants could result in mitochondrial dysfunction at later stages of life, thereby highlighting the potential health hazards of time-delayed effects of these genotoxicants in the environment.
KeywordsCaenorhabditis elegans Mitochondrial DNA damage Mitochondrial dysfunction Ultraviolet C radiation Early life exposure Genotoxicity
In recent years, potential environmental effects on mitochondrial biology have attracted increasing research interest [1, 2]. Mitochondrial DNA (mtDNA) is more sensitive than nuclear DNA (nDNA) to exposure to some chemicals, perhaps due to the absence of chromatin packing and many DNA repair pathways in mitochondria . The high lipid content of the mitochondrial membranes and the slightly negative charge of the mitochondrial matrix also attract lipophilic or positively charged compounds to mitochondria . Furthermore, non-genotoxic mitochondrial toxicants might disrupt mitochondrial function and indirectly cause mtDNA damage via generation of reactive oxygen species .
Theoretical considerations and some empirical data suggest that mtDNA damage that occurs at different stages of human life may lead to very different physiological effects. Since the quality of mitochondria in differentiated tissues depends on the quality of mitochondria in their precursors, mitochondrial damage in the early stages of human development may potentially affect mature tissue function. For example, mitochondrial toxicities exerted by developmental exposure to anti-HIV drugs in humans and laboratory models  demonstrate the importance of normal mtDNA biology during development.
The mitochondrial biology of Caenorhabditis elegans is generally similar to that of humans . The genome is 13,794 base pairs in length (Additional file 1: Figure S1), compared to 16,649 in humans. The genes encoded appear to be identical; while an atp-8 gene has not been definitively identified in C. elegans, it is probably present with an unusual sequence, as is the case in other nematodes . There are also indications that the developmental biology of mitochondria is similar in C. elegans and humans: C. elegans[7, 9, 10], like humans [11, 12], shows a large increase in mtDNA copy number with age as well as a switch from anaerobic to aerobic metabolism during development. Thus, C. elegans offers a useful model for the in vivo study of mitochondrial biology, as well as the response to toxicants .
Recent work by Furda et al.  demonstrated that persistent mtDNA damage can lead to mitochondrial dysfunction, but the response was dependent on the type of DNA damage incurred. We recently described a serial ultraviolet C radiation (UVC) exposure protocol that resulted in a large amount of irreparable mtDNA damage in C. elegans, but permitted the repair of the nDNA damage . UVC creates photodimers almost exclusively, and previous in vitro evidence [16, 17] suggests that such damage might inhibit mtDNA replication and transcription in vivo. In this work, we investigated the hypothesis that early life exposure to serial UVC results in later life mitochondrial dysfunction.
C. elegans culture and exposures
C. elegans were cultured and exposed to UVC during the L1 stage, largely as previously described . Briefly, synchronized L1 larvae were produced by overnight hatch in M9 medium following bleach-sodium hydroxide isolation of eggs as previously described . The L1 larvae were placed on peptone-free (to prevent inadvertent microbial growth) K agar plates with or without 5 μg/mL ethidium bromide (EtBr) for 48 h without food at 20°C. Half of the plates were also exposed to 7.5 J/m2 UVC radiation at 0, 24, and 48 h as described , and then transferred to OP50-seeded plates. The UVC exposure protocol is based on the fact that UVC-induced DNA damage is quickly repaired in the nuclear but not mitochondrial genome [15, 19], thus allowing for accumulation of mitochondrial DNA damage while permitting repair of nuclear DNA. This protocol results in no larval growth delay prior to the L4 stage . The transgenic strain PE255 expressing firefly luciferase as an in vivo reporter for ATP level  was generously provided by Dr. Cristina Lagido (University of Aberdeen, UK). The wildtype strain N2 was obtained from Caenorhabditis Genetics Center (University of Minnesota), which is funded by the NIH National Center for Research Resources (NCRR).
Gene expression analysis was conducted using Affymetrix /C. elegans/ GeneChip® arrays (Affymetrix, Santa Clara, CA). Hybridization targets were prepared with MessageAmp™ Premier RNA Amplification Kit (Applied Biosystems/Ambion, Austin, TX) from total RNA, hybridized to GeneChip® C. elegans Genome Arrays in Affymetrix GeneChip® hybridization oven 645, washed in Affymetrix GeneChip® Fluidics Station 450 and scanned with Affymetrix GeneChip® Scanner 7 G according to standard Affymetrix GeneChip® Hybridization, Wash, and Stain protocols (Affymetrix, Santa Clara,CA). Microarray data have been deposited in the National Center for Biotechnology Information’s GEO and are accessible through GEO series accession number GSE38997.
Microarray data preprocessing, normalization, error modeling, and initial visualization
We used Principal Components Analysis (PCA) followed by pairwise correlation analysis on unfiltered data to identify outlier samples. Data preprocessing, normalization, and error modeling were performed with Rosetta Resolver® after grouping biological replicates. The resulting fold-changes and p-values were used for Cytoscape analyses (described below).
GeneSpring-based analysis of microarray data
We used GeneSpring version GX11 (Agilent) to carry out ANOVA, PCA analysis, gene ontology (GO) enrichment analysis, and some visualization.
Interactome-based analysis of microarray data
Cytoscape (version 2.8.2) was used to overlay microarray data onto two interactomes: the high-confidence WI8 interactome compiled by the Vidal lab , and an interactome built by joining four previously-described interactomes: the integrated_function_network from the Vidal lab ; an interactome that we assembled previously  based on the BIND database  and Zhong and Sternberg  interactome; the “core” interactome described by Lee et al. ; and the higher-probability interactome generated by Alexeyenko and Sonnhammer  using a probability of functional coupling cut-off of 0.75. These networks are referred to herein as “WI8” and “Union4” and are composed of 2500 nodes and 3706 edges, and 14334 nodes and 346484 edges, respectively. The Union4 interactome is presented as Additional file 2 in .sif format. jActiveModules (version 2.23, ) was used to find, via greedy searching, the top 10 modules with a maximum overlap of 0.3, as identified at a search depth of 1 and maximum depth from start nodes of 2 (with the WI8 interactome) or 1 (with the Union4 interactome). The resulting subnetworks were analyzed for Gene Ontology enrichment with the BiNGO (version 2.44) plugin . We assessed overrepresentation using the hypergeometric test with the Benjamini and Hochberg False Discovery Rate multiple testing correction and significance level of 0.05, testing each cluster versus the entire annotation and identifying altered GO Biological Processes.
mtDNA and nDNA copy number measurements
mtDNA copy number was measured in N2 and PE255 nematodes using a modification of the real-time PCR assay described by Bratic et al.; this assay is based on a plasmid DNA-based standard curve and so generates actual rather than relative copy numbers. The only change to the assay was the use of 2-fold rather than 10-fold dilutions in the standard curve. nDNA copy number was measured using primers designed with Primer 3 : forward - 5′-GCC GAC TGG AAG AAC TTG TC-3′; reverse - 5′-GCG GAG ATC ACC TTC CAG TA-3′. These primers amplify a 164 bp region of the gene W09C5.8 (cox-4). Nuclear copy number was determined by creating a standard curve for the nuclear DNA based on young adult (24 h post-L4) glp-1 mutant nematodes raised at 25°C. At this temperature, this strain has a fixed number of cells since it has no germline proliferation  and C. elegans somatic cells do not divide in adulthood . We based the standard curve on the calculation that adults lacking germ cell proliferation would contain 3134 genomic copies [32, 33]. Real-time PCR was carried out in a 7300 Real Time PCR System (Applied Biosystems), under the following conditions: 2 min at 50°C, 10 min at 95°C, 40 cycles of 15 sec at 95°C and 60 sec at 60°C. A dissociation curve was calculated for each sample at the end of each profile. The 25 μl PCR reactions contained 12.5 μl of SYBR Green PCR Master Mix (Applied Biosystems), 8.5 μl H20, 2 μl of target-specific primers at 400 nM final concentration, and 2 μl of nematode lysate obtained as described . The ABI PRISM 7300 Sequence Detection System Software, Version 1.1 (Applied Biosystems) was used to carry out data analysis. All samples were run in triplicate and triplicates were averaged prior to analysis.
DNA damage measurements
nDNA and mtDNA damage were evaluated using a QPCR-based method as previously described  except that mtDNA damage was normalized to mtDNA copy number based on measurements obtained using the real-time method described above.
mRNA levels of five mitochondrial electron transport chain (ETC) complex subunits, including two mitochondria-encoded genes (ctb-1 and nd-5) and three nucleus-encoded genes (C34B2.8, D2030.4 and K09A9.5), were measured using real-time PCR. mRNA levels of the nuclear-encoded polg-1 gene, which encodes the C. elegans mitochondrial DNA polymerase γ, were also measured. 250 ng of mRNA isolated from C. elegans as described above was converted to cDNA using the Qiagen Omniscript Reverse Transcription kit. Real time PCR was carried out with a 7300 Real Time PCR System as described above except that the extension temperatures were 62°C for ctb-1 and nd-5, and 60°C for C34B2.8, D2030.4 and K09A9.5. The average mRNA fold change of each target gene was calculated by comparing the CT (cycle threshold) of the target gene to that of the housekeeping genes cdc-42 and pmp-3. Primers were based on the literature (ctb-1, polg-1, and nd-5 from ) except that we used an annealing temperature of 62° rather than 60°C, were designed using Primer 3, or were recommended by Dr. Marni Falk (University of Pennsylvania: C34B2.8, D2030.4 and K09A9.5). The experiment was carried out twice for a total “n” of 3–5 except when the microarray samples were used in which case the “n” was 5–7 from 5–7 experiments. Unpublished primers were as follows: cdc-42, forward - 5’- GAG AAA AAT GGG TGC CTG AA-3’, reverse - 5’-CTC GAG CAT TCC TGG ATC AT-3’ (101 bp); pmp-3, forward - 5’- GTT CCC GTG TTC ATC ACT CAT-3’, reverse - 5’- ACA CCG TCG AGA AGC TGT AGA-3’ (115 bp); D2030.4, forward - 5’- GCG AGA TGA AGG CTA CTT GG-3’, reverse - 5’-GGT GCA TTT TGG GTT TGG-3’ (115 bp); K09A9.5, forward - 5’- AGT CAT CAT CAA GGC CAT CC-3’, reverse - 5’-TTG TTG GGA TGT CAA TAC CG-3’ (185 bp); C34B2.8, forward - 5’- CTT TTC CGA AGC TTG TCT GG-3’, reverse - 5’-CTT GGC CAA CAA TTT GAG C-3’ (197 bp). All samples were run in duplicate or triplicate and replicates were averaged prior to analysis.
Steady-state ATP levels were determined by the luminescence level of the PE255 strain [20, 37]. Luminescence was measured in a 96-well microplate reader (FLUOstar OPTIMA, BMG Labtech, Ortenberg, Germany) with approximately 300 nematodes per well (in 100 μl) in the visible spectral range between 300 and 600 nm (firefly luciferase typically emits at 550–570 nm). An automated dispenser delivered 50 μl of luminescence buffer to each well, consisting of citrate phosphate buffer pH 6.5, 0.1 mM D-luciferin, 1% DMSO and 0.05% triton-X (all final concentrations). Three separate experiments with 3–5 replicates total at each timepoint were conducted.
Oxygen consumption measurement
Oxygen consumption over 2 minutes was measured in PE255 nematodes in an oxygen chamber (782 Oxygen Meter, Strathkelvin Instruments, North Lanarkshire, Scotland) as described . C. elegans were washed with K medium and counted using a nematode sorter (COPAS, Union Biometrica, Holliston, MA). Three separate experiments with 2 replicate plates per timepoint per dose each were conducted (1–4 samples per plate were measured and averaged), resulting in a total n = 4–6. Each replicate contained 1000 nematodes for the 0, 3, and 24 h time points and 500 nematodes for the 48 h time point.
All data except the microarray data were analyzed using Statview© for Windows (Version 5.0.1, SAS Institute Inc., Cary, NC). “Treatment,” “time,” and “experiment” were treated as independent variables in two- or three-way ANOVA analyses. When warranted based on initial ANOVA analyses, posthoc comparisons were carried out using Fisher’s Protected Least Significant Differences (FPLSD) test. Since oxygen consumption and ATP levels increased dramatically during larval development, and we wished to test for proportional differences based on treatment, we log-transformed those data prior to analysis. However, non-log-transformed data were plotted to avoid obscuring the large developmental changes that occurred. A p-value of less than 0.05 was considered statistically significant. Throughout the manuscript, error bars indicate the standard error of the mean.
Transcriptomic response during and 3 h after UVC exposures
We examined the transcriptomic response to a UVC exposure protocol that results in high levels of mtDNA damage but allows for repair of the nDNA damage that is also induced . Since EtBr, a specific inhibitor of mtDNA replication , exacerbates the response of C. elegans to such mtDNA damage , we also exposed half of the nematodes to EtBr. We considered the possibility that co-exposure to EtBr and UVC would lead to increased DNA damage compared to UVC alone due to photosensitization [40, 41]; however, we did not detect any difference in DNA damage with EtBr co-exposure (Additional file 1: Figure S2). In a parallel experiment, we also measured mtDNA copy number throughout the exposure. The mtDNA copy number did not change in control nematodes, nor was there a marked change in mtDNA copy number due to UVC during the exposure (Additional file 1: Figure S2).
We sampled mRNA at 4 timepoints (3 h after the first UVC exposure or “-45 h”; 1 h prior to the second exposure or −25 h; 1 h prior to the final exposure, and 3 h after the final exposure and being placed on food: Figure 1). At each timepoint, nematodes were sampled that had been exposed to UVC, EtBr, both, or neither (controls). The “n” was 4–6 samples per timepoint per treatment, each generated from different experiments (separated in time).
Most differentially expressed transcripts (DETs) were associated with time. Three-factor ANOVA (time, UVC, EtBr with Benjamini-Hochberg correction) identified 10095 genes that showed differential expression over time, using a cut-off of p < 0.001 (which results in 10 differentially expressed genes expected by chance). Only 1447 DETs resulted from EtBr treatment, and 36 DETs resulted from UVC. At this cut-off only three genes demonstrated altered responses to EtBr based on time (i.e., a time x EtBr interaction), six genes demonstrated a time-dependent response to UVC, and no genes were identified for which the response to UVC was dependent on the presence of EtBr or EtBr and time. PCA results also indicated a very strong effect of time. PCA and detailed ANOVA results for the global dataset are presented in Additional file 1: Figure S3. Since we were most interested in the combined effect of prolonged inhibition of mtDNA replication and mtDNA damage, and since the presence of food is likely to alter mitochondrial function and response to stressors, we also carried out ANOVA on the 3 h timepoint samples only. However, we found no significant (p < 0.05) UVC x EtBr interactions for any genes at 3 h.
Based on the role of mitochondrial fusion, fission, and autophagy in responding to UVC-induced mtDNA damage , we examined expression specifically of genes in these pathways. We observed a small increase in expression of some autophagy genes at −25 and −1 h, along with an inhibition of the decrease observed in many autophagy genes at 3 h, after food was available (Additional file 1: Figure S5). There was either no change or a small increase in fusion and fission genes after EtBr and EtBr +UVC, only at 3 h (Additional file 1: Figure S5).
While of less relevance for this manuscript, the transcriptomic responses to EtBr and UVC alone are interesting in their own right. The DETs (defined liberally as p < 0.05 and fold-change >1.2, based on Rosetta Resolver values) for each pairwise treatment comparison at each timepoint are provided in Additional file 3. The short-term (−45 h) response was stronger in terms of number of regulated genes for UVC than EtBr, but the reverse was true thereafter. This suggests that UVC led to a more robust signaling response, but EtBr altered more biological processes over time or that the response was slower due to the kinetics of uptake of EtBr. The most altered gene ontologies observed for UVC exposure were stress response and aging. EtBr treatment alone altered expression of many development-related genes (as described earlier) but also led to a dramatic induction of many xenobiotic metabolism genes, in particular cytochrome P450s (some of which were induced 50 to 100-fold: Additional file 1: Figure S6), but also including p-glycoprotein and glutathione S-transferase genes (Additional file 3). Some xenobiotic metabolism genes, however, were down-regulated by EtBr (e.g., cyp-35A3, ugt-37, and gst-25; Additional file 3). Finally, of all DNA repair genes that we identified previously , only one was strongly upregulated by any treatment at any time: pme-4 (Additional file 1: Figure S7).
We next carried out additional experiments to test whether mitochondrial function was in fact altered, as suggested by the transcriptomic data, and whether the mild alterations in levels of mtDNA- and nuclear genome-encoded genes would persist to later timepoints.
mtDNA damage was persistent to the L4 stage (48 h timepoint)
Measurement of mtDNA and nDNA copy number throughout development
Nuclear and mitochondrial DNA copy number in wildtype (N2) C. elegans raised ay 20 C, starting from eggs laid on k agar plates, ± standard error of the mean
nDNA copy number
mtDNA copy number (x104)
Egg, 0 h (n=6)
Hatch, 13 h (n=15)
Mid-L1, 21 h (n=7)
Mid-L2, 33.5 h (n=7)
L2/L3, 38 h (n=8)
Early L3, 41 h (n=8)
Late L3, 44 h (n=8)
L3/L4, 47 h (n=7)
Early L4, 50.7 h (n=8)
Late L4, 54.3 (n=12)
L4/young adult, 58 h (n=10)
Early young adult, 66 h (n=12)
Late young adult, 66 h (n=11)
Adult, 70 h (n=12)
UVC exposure affected mtDNA copy number but not nDNA copy number
In contrast, and as hypothesized based on the in vitro ability of UVC-induced photodimers to inhibit DNA polymerase γ , mtDNA copy number was decreased at all later timepoints (p < 0.0001 for main effects of time and treatment; p = 0.048 for interaction; timepoints past 0 h p < 0.05 UVC vs control by FPLSD; Figure 5).
These experiments were carried out in the PE255 strain, in order to permit comparison to the ATP data derived from that strain (see below). However, we observed similar results (no effect on nDNA copy number, decreased mtDNA copy number) in preliminary experiments with N2 and glp-1 strains as well (data not shown).
UVC exposure altered expression of mtDNA- and nDNA-encoded mRNAs
We also hypothesized that UVC-induced mtDNA damage would inhibit the mitochondrial RNA polymerase , resulting in a decrease in mtRNAs. Because our microarray platform has few probes for mtDNA-encoded genes, we examined those samples using RT-PCR analysis. At 3 h, but not earlier, mRNA levels for the mtDNA-encoded genes ctb-1 and nd-5 were decreased ~50% after exposure to UVC or UV + EtBr, without a change in mRNA levels for 4 nDNA-encoded mRNAs (C34B2.8, D2030.4 and K09A9.5, coding for ETC (Complex I) components, and polg-1, coding for the mitochondrial DNA polymerase γ) (Additional file 1: Figure S8). However, formal statistical comparisons of each gene at each timepoint could not be carried out due to the lack of a significant time x treatment x gene interaction.
UVC exposure resulted in a delayed decrease in steady-state ATP level
UVC exposure resulted in decreased oxygen consumption
Previous work has demonstrated that mtDNA damage generated by exposure to alkylating and oxidative agents can cause mitochondrial dysfunction in a cell culture system . However, such damage is generally repairable in mtDNA [3, 45]. In contrast, UVC-induced DNA damage is not repaired in mtDNA in C. elegans, although it can be slowly removed . Here, we report on the effect of a serial UVC exposure, which results in highly persistent mtDNA damage, on genome-level transcription and mitochondrial function.
The strong overall effect of time on our transcriptomic profiles is not surprising, since the transcriptomic response to starvation in L1s is established within 6 h, and the response to food addition is established even more quickly (by 3 h) due to RNA polymerase II accumulation in the promoters of growth-related genes . However, we were surprised not to observe a strong mitochondria-related transcriptomic response to serial UVC alone or in combination with the mtDNA intercalator EtBr. At the timepoints examined, the transcriptomic response to persistent mtDNA damage (from the UVC) and inhibition of mtDNA replication (from the EtBr) was mild and not very different than EtBr alone. Taken together, these results suggest that C. elegans lacks a way to specifically identify and respond transcriptionally to mtDNA damage, that this response is inactive at that time in development, or that any such response is very limited.
Although the transcriptomic response to EtBr by itself was quite robust, it did not include genes that are obviously related to mtDNA maintenance. Rather, the response was dominated by induction of “xenobiotic metabolism” (including cellular efflux pumps) as well as nuclear hormone receptor genes. The category of “xenobiotic metabolism,” however, should be treated with caution since the actual substrate specificity of the corresponding enzymes has generally not been tested. Nonetheless, the degree of induction is remarkable, and adds to the growing literature on the ability of this nematode to transcriptionally modulate the metabolic response to xenobiotics [47, 48, 49].
UVC, EtBr, and the combination acted to inhibit development-related transcriptomic changes associated with food addition, and also altered the mRNA levels for genes involved in energy metabolism both during starvation and after food addition. This is consistent with the induction of glycolysis genes in human cells depleted of mtDNA . It is also consistent with our other results that showed decreased ATP levels, oxygen consumption, and mtDNA copy number, since all of these typically increase during development. We note that we measured ATP levels and oxygen consumption only in PE255 nematodes, and the difference in UVC-induced mRNA levels indicates a need for caution in extrapolating results between PE255 and N2 nematodes. However, the developmental patterns that we observed were similar to those previously published for N2 nematodes, suggesting that overall mitochondrial function is likely similar in N2 and PE255 nematodes.
It is interesting that the observed decreases in mitochondrial function were not associated with a decrease in nDNA copy number. This suggests that development itself, as measured by cell division (which occurs according to an invariant pattern in C. elegans), was not significantly hindered. This conclusion is also supported by our previous observation of only a very slight delay in development at this dose of serial UVC . This is surprising given the documented ability of mitochondrial dysfunction to inhibit larval development in C. elegans[9, 51, 52]. We propose two possibilities to explain this: first, as suggested by previous work, that the developmental delay results not so much from mitochondrial dysfunction per se, but rather from a signaling event that presumably was not activated at this level of damage [52, 53]; second, that the threshold of mitochondrial dysfunction required to hinder development was not reached. These hypotheses, as well as an exploration of effects in adults, are important areas of future research.
We hypothesized that since the DNA damage was repaired in nDNA but not mtDNA, transcription of ETC components would be imbalanced, resulting in mitochondrial dysfunction. While the decreases in mRNA levels that we observed at early times were not enormous (maximally 50%), we note that in human cells, mtRNA represents between 5% and 30% of total cellular RNA , suggesting that production of high levels is important. However, there was no induction of hsp-6 and hsp-60, which respond to imbalance of ETC proteins . This suggests the possibility of retrograde signaling that permits the organism to maintain an appropriate balance of ETC proteins. Similarly, there was not a strong transcriptomic signature indicative of oxidative stress (e.g., induction of gst-4 or gcs-1: [56, 57]), as might be expected if there were significant ETC dysfunction . The lack of induction of these and other common general stress-response pathways also suggests that, although UVC is not entirely specific for nucleic acids, damage to other cellular macromolecules was not widespread. Overall, the relatively mild response to persistent mtDNA damage suggests that C. elegans has a significant ability to maintain mitochondrial function despite such damage, as we recently observed is true for primary human fibroblasts .
The significant induction in polg-1 and ETC mRNAs at 24 and 48 h post-exposure may also explain in part the nematodes’ ability to recover from damage, and to begin replenishing the mtDNA population by 48 h. It may be that polg-1 was not induced at early timepoints because of the relatively low dependence on mitochondrial function exhibited by C. elegans at those developmental stages. That insensitivity has been previously demonstrated by studies showing that mitochondrial dysfunction results in developmental arrest at the L3 or L4 stage, not earlier [9, 51, 52], and that C. elegans lacking both copies of polg-1 are able to survive to late larval stages and even in some cases early adulthood . Similarly, the induction of ETC genes supports an adaptive response, and is consistent with the ability to tolerate and transcriptionally compensate for mtDNA depletion previously observed in HeLa cells .
A fuller understanding of the basic biology of mtDNA maintenance in C. elegans will help elucidate a more complete understanding of the replicative and transcriptional response to such damage in this organism. Mitochondrial biogenesis is an important response to mitochondrial stress or dysfunction in mammalian cells , and our mRNA induction data supports such a response in C. elegans. However, mitochondrial biogenesis per se has not been described in C. elegans. In addition, although there are many similarities with mammalian mtDNA, there are also differences. For example, TFAM has not been found in C. elegans—perhaps because if present, it may not have a transcriptional role, as appears to be the case in yeast . It is also interesting that in C. elegans there are apparently no genes on the light strand, raising questions about how transcription and replication are coupled in this species, since light strand transcription is the mechanism for priming mtDNA replication in humans [63, 64].
In summary, our results support the hypothesis that early life exposure to persistent mtDNA damage can lead to later life mitochondrial dysfunction. However, they also highlight the ability to compensate for or respond to such damage in vivo. Some of this capacity is likely the result of the ability to clear such damage via mitochondrial dynamics and autophagy . An important direction of future research will be to investigate how deficiencies in those processes—which are observed in the human population—will affect the response to such damage.
We thank Marni Falk for primer information, Alexandra Trifunovic for a plasmid-based mtDNA copy number standard curve, and Bernard Lemire for advice regarding oxygen consumption measurement. We thank Margaret Gustafson, Shawn Ahmed, and Jonathan Freedman for their advice and assistance in this study. This work was funded by NIEHS and NINDS (1P30 ES011961, 1R01-ES017540, and 1R21 NS065468 to JNM), the Society of Toxicology (Colgate-Palmolive Awards for Student Research Training in Alternative Methods to MCKL); American Foundation of Aging Research (GlaxoSmithKline Foundation Award to MCKL).
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