Prenatal hyperbaric normoxia treatment improves healthspan and regulates chitin metabolic genes in Drosophila melanogaster

Characterization of HN in D. melanogaster development

To determine the effects of HN in D. melanogaster, we examined whether prenatal HN exposure affected development at each stage of growth. We observed no significant effects of HN on growth compared to controls. Although the timing from larva to pupa was delayed at day 5 (p =0.003), the total larval growth pattern, total development time from egg to eclosion (Fig. 1A) and total average eclosion rate (egg to adult survival) did not differ between HN and control groups (Fig. 1B). Moreover, HN did not affect the mean body weight of males, females or larvae at eclosion (Fig. 1C) or wing size (Fig. 1D).

Figure 1. The effects of HN on growth pattern and rate. (A) HN-treated larvae pupariated at normal time points and were slightly delayed at 5day after egg laying (>80 animals/group). (B) The egg-to-adult viability was no significant difference between control and HN exposure (>60 eggs/group, n≥5 independent experiments). (C) Average weight of larvae (Ctrl, n=187; HN, n=192) and adult flies (Ctrl male, n=53; HN male, n=54; Ctrl female, n=41; HN female, n=46) indicated. (D) HN-treated flies had no significant change in wing size (5 flies/group, n≥3). Error bars denote the standard error of the mean (SEM). NS: not significant.

Protection against heat and oxidative stresses by prenatal HN exposure

We previously showed that hyperbaric normoxia protects normal fibroblasts (WI-38) against oxidative stress [16]. We therefore examined whether HN treatment also enhanced stress tolerance in D. melanogaster (Table 1A). To determine the tolerance to oxidative stress, flies were fed 18 mM paraquat added to standard food and their survival was monitored. We found that for both male and females, HN-treated flies exhibited a significant resistance to oxidative stress, 12% increase of median survival in males (Fig. 2C) and 47% in females (Fig. 2F), whereas control flies exhibited high sensitivity to paraquat. Under 40°C heat stress, both male and female HN-treated flies showed increased thermal tolerance by 20% in both males (Fig. 2A) and females (Fig. 2D). In addition, when subjected to nutrient deprivation, only female flies showed increased resistance to starvation, compared to their controls (males, Fig. 2B; females, Fig. 2E). These results suggest that HN plays a protective role against different stresses in Drosophila, which is in agreement with previous in vitro reports [16].

Figure 2. Enhanced heat and oxidative stress tolerance by prenatal HN exposure in both sex. HN exposure led to significantly increased resistance to heat stress in male (A) p =0.0001; female flies (D) p =0.021 and oxidative stress (C) male, p =0.031; (F) female, p =0.0004. HN exposure affected starvation resistance in female (B) p <0.0001) but not in male flies (E) p =0.2004). HN-treated flies presented a significant increased (black: controls; grey: HN exposure).

Enhanced longevity and healthspan by hyperbaric normoxia

To gain further insight into the protective effects of HN, we assayed the lifespan in treated and untreated flies (Table 1B). We found that the median lifespan of HN-treated flies was increased by 12% in males (Fig. 3A) and by 14% in females (Fig. 3B). Motor performance is regarded as a marker of healthspan in D. melanogaster [28,29]. To measure this, we tested the vertical climbing ability of treated and untreated flies. We found that HN treatment improved the flies’ ability to reach the top of the vial (males, Fig. 3C; females, Fig. 3D), indicating an improved motor performance. We also tested reproductive output, which is often negatively correlated with extended lifespan and healthspan [30,31]. However, our results showed that while HN-treated female flies showed decreased fertility at day 8, there was no detrimental effect on average daily fecundity compared to controls (Fig. 3E). This implies that prenatal HN exposure has the ability to delay the physiological age without any associated reproductive costs.

Figure 3. Increased healthspan in HN-treated flies. HN exposure led to increased mean lifespan by 12% in males (A) p <0.0001) and by 14% in females (B) p <0.0001. HN-treated flies showed a significant higher climbing performance then control groups (C) male, p =0.0007; (D) female, p = 0.028. (E) HN exposure did not affect the fecundity while there was decreased fertility at day 8 (p =0.009). Error bars denote SEM. Values with different superscripts are significantly different at p <0.05 (* = p <0.05, ** = p <0.01, *** = p <0.01).

Changes in ROS production and oxidative stress-related gene expression

Aging leads to increasing ROS as well as impaired motor performance associated decreased lifespan [32]. The relationship between ROS and healthspan has been studied extensively. In the present study, we examined ROS levels in HN-treated flies, using a FOX assay. Compared to control flies, the average amount of ROS in HN-treated flies was decreased by 10% for both males and females (Fig. 4A). Otherwise, there was no difference in expression levels of mitochondrial-encoded genes, such as cytochrome c oxidase subunits I and III (COXI, COXIII) and cytochrome b (Cytb) in males or females (Fig. 4B). To test if ROS reduction by HN exposure leads to altered gene expression associated with aging and oxidative stress, we monitored mRNA levels of candidates. We analyzed the gene expression level of Drosophila methushelah (mth) and Sir2, 4E-BP, FOXO and Tor in nutrient signaling, there was no changes (males, Fig. 5A; females, Fig. 5B). However, Oxidative stress-related genes including hsp70, catalase, glutathione synthase (GS), Jafrac 1, and Superoxide dismutase 2 (MnSOD) were up-regulated following HN exposure (males, Fig. 5C; females, Fig. 5D). These data suggest that the prolonged healthspan that results from prenatal HN exposure is associated with anti-oxidant defense mechanisms.

Figure 4. Decreased ROS production by HN exposure. (A) ROS generation was reduced in HN-treated flies (male, p =0.013; female, p =0.035). (B) The expression levels of mitochondrial DNA were not changed in both sex. Error bars denote SEM. * = p <0.05, ** = p <0.01, *** = p <0.01.

Figure 5. Altered oxidative stress-related gene expression by HN exposure. (A-B) Aging related gene levels were not changed. (C-D) HN-treated mRNA expression related with oxidative stress genes were increased in both sex. Error bars denote SEM. * = p <0.05, ** = p <0.01, *** = p <0.01.

Global gene expression profiles in HN treated healthspan increased flies

To obtain a global view of the genomic response to HN treatment in the healthy aging phenotype in male and female flies, we performed a microarray using 32,162 probe sets and analyzed it together with its control. Genes with a more than 1.5-fold change (with statistical significance) following HN treatment were presented as Venn diagrams (Fig. 6A). In total, 4,293 genes (3,946 induced; 347 repressed) were altered in males and 1,841 genes (1,163 induced; 678 repressed) were altered in females following prenatal HN exposure. Comparison of these two sets revealed a significant overlap of 473 genes, among which 441 genes were up-regulated in the HN-treated group. These up-regulated genes can be analyzed in Gene Ontology (GO) which is grouped into three major categories, including biological process (BP), cellular component (CC), and molecular function (MF) (Fig. 6B). The top five sub-categories in BP were indicated in aminoglycan metabolic process (18 genes), chitin metabolic process (17 genes), Polysaccharide metabolic process (7 genes), myofibril assembly (7 genes) and ciliary or flagellar motility (5 genes).

Figure 6. Differential gene-expression analysis and enriched gene ontology categories. (A) Venn diagram comparing the up-regulated and down-regulated genes in HN-treated flies. (B) Annotated genes from the overlap of up-regulated gene lists can be divided into three major categories, including biological process (BP), cellular component (CC), and molecular function (MF). The vertical scale on the left indicated the number of genes in the sub-categories. (C) The functional enrichment map were illustrated for a number of biological function including the enriched target genes.

Chitin metabolism is involved in the improved healthspan seen in HN-treated flies

Our microarray analysis revealed changes in 18 transcripts involved in aminoglycan metabolic process. Among these, 17 transcripts were found to be involved in chitin metabolism, which is related to aminoglycan metabolism according to functional enrichment map. Using KEGG pathway analysis to confirm the overlapping up-regulation following prenatal HN exposure, amino sugars, and nucleotide sugar metabolism were enriched in the HN-treated group. Potential healthspan-promoting genes Chitinase 6 (Cht6: CG43373), Chitinase 7 (Cht7: CG1869) and kkv, were selected for further analysis. Validation by qPCR revealed that mRNA levels of Cht6, Cht7, and krotzkopf verkehrt (kkv) were highly increased in prenatal HN-exposed flies (males, Fig. 7A; females, Fig. 7B). These observations identify chitin metabolism as a potential molecular mechanism involved the hormetic effects of HN that result in a prolonged healthspan.

Figure 7. Quantitative real-time PCR validation of HN-treated healthspan genes acquired from microarray analysis. Long-lived HN exposure flies increased the expression of 3 genes from the most up-regulated genes among GO terms. Error bars denote SEM. Values with different superscripts are significantly different at p <0.05 (* = p <0.05, ** = p <0.01, *** = p <0.01).

Fly stocks and maintenance

Wild-type Drosophila melanogaster w¹¹¹⁸ were obtained from the Bloomington Drosophila Stock Center (Bloomington, IN, USA) and maintained in a climate-controlled incubator with 25°C and 50 ± 5% relative humidity on 12:12 h light/dark cycle. All flies used in this study were raised on fresh medium containing 6% cornmeal, 3% yeast, 3% sugar, 6% glucose, and 0.6% agar.

Experimental design for hyperbaric normoxia incubation

All hyperbaric normoxia (HN; 2ATA in normoxic conditions) exposures were carried out in a custom-made animal incubator designed for pressure control [55] without change of temperature (25°C), humidity (50 ± 5%) and under a 12:12 h light/dark cycle. Fertilized eggs from 30 3-day-old pairs were collected in batches of 30 into vials, then exposed to HN until eclosion. After HA exposure all newly born (F1) flies were maintained in a climate-controlled incubator as described above.

Measuring developmental time, eclosion rate and fecundity

We measured larval development time from egg to pupa (at 24-h intervals), eclosion rate from egg to adult and weight of larvae and adults (male or female). For fecundity, newly eclosed F1 generations of 15 virgin female and 7 male flies were collected and placed into a vial. Flies were transferred to fresh vials every day and the number of eggs laid in a 24-h period was counted for 15 days with ten replicates.

Body weight and wing size analysis

Body weight of individual larva and adult flies was measured. Flies were reared under the same growth conditions and were age-matched (2 days old) before weighing.

To estimate body size, variation in wing size was measured. The left wing was removed and mounted onto double-sided tape on glass slides and sealed with a cover glass. Images of the wings were taken using a Leica MZ 125 microscope and Leica Application Suite LAS version 3.7.0 software (both from Leica Microsystems).

Climbing and longevity assays

The climbing assay was performed as previously described by Ganetzky et al. [28] with some modifications. Ten-day-old flies (20 males or 20 females per vial) were collected and placed into a vial (height, 10 cm). After a 1 h recovery period from anesthesia, each vial was gently taped to the bottom. At this point, we counted the number of flies that climbed up to a vertical distance of >7.5 cm within 5 s or 7 s. This procedure was repeated five times. After five trials, the number of flies in each vial was counted. At least 100 flies were used for each condition tested.

Lifespan assay was performed as described previously [56]. In short, 80 male or female flies (1-3 day after eclosion) were cultured in an aging chamber [57]. The number of dead flies was counted every 2–3 days, when fresh food vials were replaced. This procedure was repeated three times.

Assays for stress resistance of adults

To examine ROS tolerance, flies were placed in a vial containing fresh food and 18 mM paraquat (methyl viologen; Sigma-Aldrich, USA), after wet-starvation in a vial containing 1% agar for 6 h. The number of dead flies was recorded every 3 h until all flies had died. Similarly, starvation resistance was measured by feeding with only water in a vial containing 1% agar (Sigma-Aldrich, USA). Dead flies were counted every 3 h. To test heat tolerance, flies were kept in a 40°C incubator and paralyzed flies were counted every 10 min. Each experiment was performed with 10-day-old flies (20 males or 20 females per vial) with at least three replicates.

Quantitative real-time PCR analysis

F1 generation flies were frozen in liquid nitrogen and stored at -80°C until analysis. Total RNA was extracted from homogenized whole-body lysates using RNAiso reagent (TAKARA, Japan). After treating with DNase I (Invitrogen, USA), cDNA synthesis was performed using a PrimeScript RT Reagent Kit (TAKARA, Japan). Quantitative real-time PCR was performed using Power SYBR Green (Applied Biosystems, USA) and StepOnePlus Real-Time PCR System (Applied Biosystem, USA). To determine mRNA expression levels for each gene, C_T values were normalized to those of rp49. Mean fold induction was calculated from the values from 3-6 independent experiments.

Quantification of mtDNA

Total DNA was extracted from 10 day old flies from the F1 generation using a G-spin Genomic DNA extraction kit (iNtRON Biotechnology, Korea) and subjected to quantitative real-time PCR as described above. Copy number of mtDNA was estimated using Cox I, Cox III, and Cyt b primers [58], relative to rp49.

ROS measurement

The rate of ROS production in 20 ten day old flies was measured using a ferric-xylenol orange peroxide assay [59]. Whole bodies were minced in 250 µl distilled water containing aminotriazol (2 mg/ml). Experiments were performed in triplicate. After centrifugation 40 µl of supernatant was mixed with 360 µl FOX reagent (250 µM Fe(NH₄)2SO₄·6H₂O, 25 mM H₂SO₄, 4 mM butylated hydroxyl toluene in HPLC grade methanol, 100 µM xylenol orange) containing 100 mM sorbitol (all from Sigma-Aldrich, USA). After incubation at room temperature for 40 min, absorbance of the ferric-xylenol orange complex was estimated at 560 nm using a SpectraMax microplate reader (Molecular Devices, USA).

Microarray

Total RNA from 20 male and 20 female flies from control and HN-treated groups were collected as described above. For each RNA, the synthesis of target cRNA probes and hybridization were performed using a LowInput QuickAmp Labeling Kit (Agilent Technologies, USA) according to the manufacturer’s instructions. Briefly, 25 ng total RNA was added to the T7 promoter primer mix and incubated at 65°C for 10 min. cDNA master mix (5X First strand buffer, 0.1 M DTT, 10 mM dNTP mix, RNase-Out, and MMLV-RT) was added to the reaction mix. Samples were incubated at 40°C for 2 h, and RT and dsDNA synthesis was terminated by incubating at 70°C for 10 min. Transcription master mix was prepared according to the manufacturer’s protocol (4X Transcription buffer, 0.1 M DTT, NTP mix, 50% PEG, RNase-Out, inorganic pyrophosphatase, T7 RNA polymerase, and Cyanine 3-CTP). Transcription of dsDNA was performed by adding the transcription master mix to the dsDNA reaction samples and incubating at 40°C for 2 h. Amplified and labeled cRNA was purified on an RNase mini column (Qiagen, USA) according to the manufacturer’s protocol. Labeled cRNA target was quantified using an ND-1000 spectrophotometer (NanoDrop Technologies, USA). After checking labeling efficiency, 1650 ng of each cyanine 3-labeled cRNA target was used for cRNA fragmentation by adding 10X blocking agent and 25X fragmentation buffer and incubating at 60°C for 30 min. Fragmented cRNA was resuspended with 2X hybridization buffer and directly pipetted onto an assembled Agilent Drosophila (V2) Gene Expression 4X 44K Microarray. Arrays were hybridized for 17 h at 65°C in an Agilent Hybridization oven (Agilent Technologies, USA). Hybridized microarrays were washed according to the manufacturer’s protocol (Agilent Technologies, USA).

Microarray data acquisition and GO analysis

Hybridization images were analyzed using an Agilent DNA Microarray Scanner (Agilent Technologies, USA) and data quantification was performed using Agilent Feature Extraction software 10.7 (Agilent Technologies, USA). Average fluorescence intensity was calculated for each spot and local background was subtracted. All data normalization and selection of fold-changed genes were performed using GeneSpringGX 7.3.1 (Agilent Technologies, USA). Normalization for the Agilent one-color method was performed: Data transformation, set measurements less than 5.0 to 5.0; Per Chip, normalize to 50th percentage. Averages of normalized ratios were calculated by dividing the average of control normalized signal intensity by the average of test normalized signal intensity.

Functional annotation of genes was performed according to DAVID annotation tools [60,61]. Default DAVID parameters were used. Venn diagrams from two gene list (male and female) in microarray data analysis were created by R software package. All raw data were deposited in the NCBI Gene Expression Omnibus under accession number GSE84069.

Statistical analyses

Statistical analyses were conducted using PRISM version 6 (GraphPad Software, Inc., USA). All data except survival curves were presented as mean ± SEM and were statistically evaluated using Student t-test distribution. Results of lifespan and stress resistance studies were analyzed using the Kaplan-Meier survival test. Comparisons among groups were conducted using Gehan’s Wilcoxon test.