Ethics Statement

Written informed consent was obtained from all the patients (from the next of kin, caretakers, or guardians on the behalf of the minors/children patients) in accordance with the Declaration of Helsinki. The study was approved by the Shanghai Rui Jin Hospital Review Boards. Animals were used according to the protocols approved by the Shanghai Rui Jin Hospital Animal Care and Use Committee.

Patients

Two hundred and four patients with lymphoma [103 diffuse large B-cell lymphoma (DLBCL) and 101 T-cell lymphoma (TCL) cases], 127 men and 77 women aged 14 to 82 years were included in this study. Histological diagnoses were established according to the World Health Organization classifications. Frozen tumor specimen was available in 114 lymphoma patients and 40 age- and sex-matched reactive hyperplasia cases.

Cell Lines and Reagents

The B-lymphoma cell line Namalwa and T-lymphoma cell line Jurkat were obtained from American Type Culture Collection. Hydroquinone (Sigma-Aldrich) was dissolved in normal saline before use. Benzo(a)pyrene (BaP, Sigma-Aldrich) was dissolved in dimethyl sulfoxide (DMSO) as a stock solution of 400 mg/ml.

Cell Proliferation and Flow Cytometric Assay

Cell growth was measured by MTT assay and cell proliferation was determined by 5-ethynyl-2′-deoxyuridine (EdU) incorporation assay using Cell-Light EdU kit (Ribobio Co., Ltd., China) at 48 h. Cell cycle and cell apoptosis were assessed at 48 h as previous reported [7].

Genome-wide Copy Number Variation (CNV) Analysis

Genomic DNA was extracted using Wizard Genomic DNA Purification Kit (Promega). Genome-wide CNV genotyping was performed on frozen tumor samples of 25 DLBCL, 20 TCL and 8 reactive hyperplasia cases, using Human 610-Quad_v1 (610 k SNP probes) or 660 W-Quad_v1 (660 k SNP probes) DNA Analysis BeadChips. Regions were determined based on the Log R Ratio (LRR) of the signal intensity and B allele frequency (BAF) of genotyping call from the sample using platform of GenomeStudio V2011.1 with CnvPartition 3.1.6 (Illumina). All the data is available on NCBI (Accession number GSE47357).

GSTT1 Genotyping and Expression

GSTT1 deletion was detected on frozen tumor (114 cases) and peripheral blood (the rest 90 cases without frozen tumor specimen) of lymphoma patients by multiplex polymerase chain reaction (PCR) method, using albumin gene as an internal positive control, as previously reported [8]. The normal control group comprised 205 unrelated healthy volunteers. Blood samples were collected and leukocytes were isolated after hypotonic lysis of erythrocytes. Genomic DNA was extracted and amplified using the primers: GSTT1∶5′-TTCCTTACTGGTCCTCACATCTC-3′ and 5′-TCACCGGATCATGGCCAGCA-3′, and albumin:5′-GCCCTCTGCTAACAAGTCCTAC-3′ and 5′-GCCCTAAAAAGAAAATCGCCAATC-3′.

Total RNA was extracted using Trizol reagent and reverse-transcribed using PrimeScript RT reagent Kit with gDNA Eraser (TaKaRa). Real-time PCR was performed on frozen samples of lymphoma and reactive hyperplasia patients, using ABI PRISM 7900HT and specific probes for GSTT1 (Assay ID: Hs01091675_g1) and GAPDH (Life Technologies). A relative quantification was calculated using the 2^−ΔΔCT method.

Gene Network and Pathway Analysis

Human Genome U133 Plus 2.0 Array GeneChip microarray (Affymetrix) was performed on tumor samples of 8 DLBCL patients and analyzed by Expression Console software (Partek GS 6.5, Affymetrix). The data is available on NCBI (Accession number GES47355). Human LncRNA Microarray V2.0 (Arraystar Inc.) was performed on tumor samples of 8 TCL cases and analyzed by Agilent Feature Extraction Software (Agilent Technologies).

Genes were subsequently filtered by comparing their expression levels between the GSTT1-deleting and non-deleting patients. Statistical differences were calculated and the genes with P<0.05 were analyzed for enrichment of KEGG pathways using Database for Annotation, Visualization and Integrated Discovery (DAVID v6.7, http://david.abcc.ncifcrf.gov) for network composition analyses. Genes of pathway(s) significantly involved in both DLBCL and TCL were hierarchical clustered using MeV v4.8.1 (Dana-Farber Cancer Institute).

GSTT1 Transfection

GSTT1 expression vector (GSTT1, NM_000853.2) and the negative control vector (FU, pReciever-M46) were obtained from GeneCopoeia. The recombinant lentivirus vector PGC-GSTT1-IRES-GFP-LV and PGC-FU-GFP-LV were produced and packaged by co-transfecting 293T cells with the package vector. The supernatant of 293T cell culture were condensed and the virus titers were approximately 3×10⁹ TU/ml. To transfect Namalwa and Jurkat cells, the multiplicities of infection were 50 and 10, respectively.

Comet Assay

DNA damage was determined by the comet assay with Reagent Kit for Single Cell Gel Electrophoresis Assay (Trevigen, Inc.). In addition to frank DNA strand breaks, oxidised bases were measured by conversion to breaks using endonuclease III (recognizing oxidised pyrimidines) or formamidopyrimidine DNA glycosylase (FPG, specific for oxidised purines). Measurements of comet parameters % DNA in the tail, tail length and tail moment were obtained. Net enzyme-sensitive sites were calculated by subtracting the comet score after incubation with buffer alone from the score with enzyme.

Tissue Array

A human lymphoma tissue array (NHL482) was obtained from US Biomax, Inc. GSTT1 and p53-binding protein 53 BP1 expression were scored semi-quantitatively based on staining intensity and distribution using the immunoreactive score, as previously reported [7].

Immunohistochemistry and Immunofluorescence Assay

Immunohistochemical analyses were carried out on 5-µm-paraffin sections or acetone-fixed cells with an indirect immunoperoxidase method using the antibodies against GSTT1, 53 BP1 (Abcam), Ki67 (Dako) and MYC (Abcam). Immunofluorescence assay was performed on acetone-fixed cells using mouse anti-human-γH2AX antibody followed by donkey anti-mouse-IgG antibody, as well as rabbit anti-human-53 BP1 and rabbit anti-human-pCHK1 antibody followed by donkey anti-rabbit-IgG antibody (Abcam). Nuclei were counterstained with DAPI.

Western Blot

Western blot was performed as previously described [7]. Actin (Sigma) and LaminB (Abcam) were used to ensure equivalent loading of total and nuclear protein, respectively. Antibodies against GSTT1 and CHK1 were obtained from EPITOMICS. Anti-pCHK1, pCHK2 and CHK2 antibodies were from Cell Signaling. Anti-MYC and 53 BP1 antibodies were from Abcam. Horseradish peroxidase-conjugated goat anti-mouse-IgG and goat anti-rabbit-IgG were from Santa Cruz Biotechnology Inc.

Tumorigenicity Assay in Murine Models

Five-week-old female BALB/c nude mice were obtained from Shanghai Laboratory Animal Center. Mice were injected subcutaneously into the right flank with lymphoma cells. For each cell line, mice were divided into 3 subgroups. Namalwa cells were injected with 1×10⁷, 2×10⁶ and 5×10⁵, and Jurkat cells were injected with 4×10⁷, 1×10⁷ and 2×10⁶, respectively. The number of the tumors formed was determined until 4 weeks after injection. Then mice were sacrificed, with tumor tissue samples fixed in formaldehyde and further processed for paraffin embedding.

Cloning and Plasmid Construction in Zebrafish

Adult zebrafish (Danio rerio) were maintained following established guidelines [9] at 28°C on a 14 h:10 h light:dark cycle. Zebrafish gstt1a and gstt1b genes were identified based on homology to human GSTT1. The specific primers were designed according to genomic sequence in the UCSC data base (University of California, Santa Cruz) to amplify part of gstt1a and gstt1b genes: gstt1a:5′- CGGGATCCGGCGCCTCTCTTCTTTTCTT-3′ and 5′-CCCTCGAG CTGCATCATCTCCACAATGG-3′; gstt1b:5′-CGGGATCCTGATCCCAAAGGGTTGAGTC-3′ and 5′-CCCTCGAGGAACAAAAAGGTGCCATTCG-3′; gstt1a-EGFP reporter:5′- GATCCACTTACTTCCACAATGCCGCTGGAGCTGTATCTCGATCTGCATTCCCAGCCG-3′ and 5′- AATTCGGCTGGGAATGCAGATCGAGATACAGCTCCAGCGGCATTGTGGAAGTAAGTG-3′; gstt1b-EGFP reporter:5′- GATCCCTTTTTAAACATGACTCTGGAAATTTACTTGGACCTGTTTTCCCAGCCCTGG-3′ and 5′- AATTCCAGGGCTGGGAAAACAGGTCCAAGTAAATTTCCAGAGTCATGTTTAAAAAGG-3′; myca:5′-CGGGATCCTTTCCAAGAACTCCCACCCC-3′ and 5′-CCCTCGAGTTTAATCTAGGGCTGCGCAG-3′. The amplified PCR product was purified and subcloned into pCS2⁺ for subsequent in vitro synthesis of mRNA.

Whole-mount in situ Hybridization (WISH) in Zebrafish

To localize gstt1a and gstt1b in zebrafish, whole-mount in situ hybridization (WISH) was carried out. pCS2⁺ containing part of gstt1a, gstt1b, rag1 or myca (729 bp, 473 bp, 1000 bp and 231 bp, respectively) was utilized to generate antisense RNA probes for gstt1a, gstt1b, rag1 and myca using digoxigenin-11-uridine 5′-triphosphate (Roche Applied Science). Zebrafish embryos were fixed in 4% paraformaldehyde at the stages indicated and WISH was performed as previously described [10].

Microinjection of Zebrafish Embryos

The gstt1a and gstt1b morpholino oligonucleotides (MO) and its 5 bp-mismatch control were synthesized by Gene Tools LLC. The sequences were as follows: gstt1a:5′-CGAGATACAGCTCCAGCGGCATTGT-3′; 5 bp-mismatch of gstt1a: 5′-CGAcATAgAcCTCCAcCcGCATTGT-3′; gstt1b:5′- GGTCCAAGTAAATTTCCAGAGTCAT-3′; 5 bp-mismatch of gstt1b:5′- GGTCgAAcTAAATTTgCAcAcTCAT -3′. MOs were diluted to 1 mM stock solution in 1×Danieau buffer and were microinjected at a volume of 2 nl into one-cell stage embryos using an air pressure injector and glass capillaries. Injection experiments were performed in triplicate.

The vectors pCS2⁺-MO-gstt1a and pCS2⁺-MO-gstt1b were linearized with XhoI and transcribed in vitro with SP6 RNA polymerase in the presence of m7G (59)ppp(59)G (Ambion) to produce capped transcripts and were microinjected into one-cell stage embryos for MO efficiency evaluation.

Transmission Electron Microscopy

The embryos of zebrafish were fixed overnight with 2% glutaraldehyde/0.1 M phosphate-buffered saline (pH 7.3) at 4°C. The embryos were then post-fixed in 1% osmium tetroxide at 4°C for 1 h, rinsed thoroughly with distilled water, dehydrated by graded ethanol and freeze-dried. The samples were sputter-coated in Epon812 (TAAB Laboratories) and ultrathin sections were prepared, stained with uranyl acetate and lead citrate, and examined with a PhilipsCM120 transmission electron microscopy (Philips).

Statistic Analysis

Assays were set up in triplicate and the results were presented as Mean±S.E. Variance between the experimental groups was determined by two-tailed t-test or Mann-Whitney test. The logistic regression test was used to compare the differences in genotype frequencies between patients and controls adjusted by sex and age. Differences were considered significant when the 2-sided P<0.05.

GSTT1-null Genotype is Frequently Observed in Lymphoma Patients

A homozygous loss in chr22q11.23 spanning GSTT1 gene (22,706,139 bp to 22,715,284 bp, NCBI Build 36) was detected in 15/25 (60%) of DLBCL and 7/20 (35%) of TCL patients, comparing with 1/8 (12.5%) of reactive hyperplasia cases (Figure 1A). Figure 1B depicted data for the region of 22,500,000 bp to 22,900,000 bp of chromosome 22. In the normal control, the LRR was distributed around zero corresponding to DNA copy number 2, whilst the BAFs were clustered around values of 0, 0.5 and 1 that correspond to the diploid genotypes AA, AB and BB. The GSTT1-null genotype presented a much more complex scenario with extensive genomic rearrangements leading to considerable variation in the SNP data.

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Figure 1. GSTT1-null genotype is frequently observed in patients with lymphoma.

A: GSTT1 deletion detected by copy number variation (CNV) analysis (upper panel) and validated by semi-quantitative PCR (lower panel) in diffuse large B-cell lymphoma (DLBCL, 15/25, 60.0%), T-cell lymphoma (TCL, 7/20, 35.0%) and reactive hyperplasia (1/8, 12.5%). B: SNP data illustrating the distribution of B allele frequencies (BAF) and Log R Ratio (LRR) values across the region of chromosome 22 (22,676,385 bp to 22,717,669 bp) in the GSTT1-deleting cases and normal controls. C: Semi-quantitative PCR validation of GSTT1 deletion in expanded sample-set of DLBCL (57/103, 55.3%), TCL (55/101, 54.5%) and normal controls (86/205, 42.0%). *P<0.05 comparing with normal controls.

https://doi.org/10.1371/journal.pone.0089302.g001

Using semi-quantitative PCR, GSTT1 genotypes were further investigated. GSTT1 gene deficiency was deduced from the absence of the specific 460 bp fragment. Presence of a 350 bp albumin fragment confirmed proper functioning of the method (Figure 1A). Of the 204 patients, 112 cases (54.9%) were GSTT1-null. Prevalence of the GSTT1 deletions was 55.3% (57/103) in DLBCL and 54.5% (55/101) in TCL, significantly higher than that in normal controls (86/205, 42.0%, P = 0.027 and P = 0.034, respectively, Figure 1C), adjusted by sex and age. This translated into an increased risk of lymphoma for individuals with the GSTT1-null genotype (Odds ratio = 1.698; 95% confidence interval = 1.145–2.518).

GSTT1 Deletion Results in Loss of Protein Expression

GSTT1 gene expression was measured by real-time quantitative PCR in 114 patients with frozen tissue specimen. GSTT1-deleting patients presented significant downregulation of GSTT1 gene expression in both DLBCL and TCL, comparing with those without deletion (P both <0.001, Figure 2A). GSTT1 protein was detected by tissue array. GSTT1 was negative in 56.0% (14/25) of DLBCL and in 50.0% (4/8) of TCL, respectively, in accordance with the percentage of GSTT1 deletion in the two subtypes (Figure 2B). Moreover, 53BP1 was more frequently expressed in GSTT1-negative cases than in GSTT1-positive cases [DLBCL, 85.7% (12/14) vs 45.5% (5/11), P = 0.032, T-NHL, 100% (4/4) vs 0% (0/4), P = 0.028].

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Figure 2. GSTT1 deletion is related to decreased gene and protein expression and enhanced cell cycle progression.

A: GSTT1 gene expression assessed by quantitative real-time PCR in the GSTT1-deleting patients and normal controls. B: GSTT1 protein expression detected by tissue array. C: Geneset of cell cycle related proteins revealed by gene network and pathway analysis on microarray data of DLBCL and TCL.

https://doi.org/10.1371/journal.pone.0089302.g002

Gene expression profile was assessed by microarray in frozen tissue sample of 8 DLBCL and 8 TCL patients with the history of PAH exposure (four each for GSTT1-deleting and non-deleting cases). The pathway identified with statistical significance by the differentially expressed GSTT1 revolved around key proteins of cell cycle progression (Figure 2C), indicating that GSTT1 deletion is biologically functional and relevant to lymphoma cell proliferation.

GSTT1 Protects Lymphoma Cells from PAH-induced DNA Damage

To further elucidate its biological function in lymphoma, GSTT1 gene was transfected to GSTT1-negative Namalwa and Jurkat cells. Comparing with the negative control (FU), lymphoma cells expressing GSTT1 (GSTT1) showed increased levels of gene and protein expression, as revealed by semi-quantitative PCR (Figure 3A) and by immunostaining assay (Figure 3B), respectively.

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Figure 3. GSTT1 expression protects lymphoma cells from PAH-induced DNA damage.

A: GSTT1 gene expression assessed by semi-quantitative PCR in Namalwa and Jurkat cells transfected with GSTT1 (GSTT1) and the negative control vector (FU). B: Images represent results from three independent experiments. GSTT1 protein expression detected by immunohistochemistry assay. C: DNA damage measured by alkaline and modified comet assay in Namalwa and Jurkat cells treated with Hydroquinone (Upper panels). Mean tail moments were calculated in the same cells (Lower panels). Data represents Mean ± SE from at least 50 cells in each group. D: Immunofluorescence assay of γH2AX and 53BP1 in Hydroquinone-treated lymphoma cells. *P<0.05 comparing with the FU cells.

https://doi.org/10.1371/journal.pone.0089302.g003

Hydroquinone, common metabolites of PAH [11], was used to treat lymphoma cells. Fifty percent of growth inhibition (IC50) was first measured in both cell lines at concentrations ranging from 1.25 to 40 µM. The IC50 value was 14.4 µM in Namalwa cells and 18.7 µM in Jurkat cells, respectively. To mimic the environmental exposure status, lymphoma cells were then cultured with low-dose Hydroquinone (at a concentration of approximately 10% of IC50 with minimal cytotoxicity, 1 µM for Namalwa GSTT1/FU cells and 1.5 µM for Jurkat GSTT1/FU cells, respectively) in a repeat manner [12]. Briefly, cells were treated once a week with Hydroquinone for 24 h and weekly for 4 times in total. Normal saline was referred as the solvent control.

DNA strand breaks were assessed using comet assay, and in addition DNA oxidation damage, namely oxidised pyrimidines and purines, using lesion specific enzymes Endo III and FPG. As manifested by increased parameters % DNA in the tail and prolonged tail length, DNA damage was less intensive in Hydroquinone-treated GSTT1-expressing lymphoma cells (GSTT1) than in the negative control cells (FU). Tail moment was further assessed by parameters % DNA×tail length. The results showed that tail moment, as well as EndoIII- and FPG-sensitive sites, were significantly reduced in the GSTT1 groups than in the FU groups (Namalwa, P = 0.037, P = 0.042 and P = 0.042, Jurkat, P = 0.041, P = 0.049 and P = 0.049, respectively, Figure 3C).

γH2AX and 53 BP1 are sensitive markers of DNA damage [13]. As detected by immunofluorescence assay, FU cells presented increased nuclear levels of γH2AX and 53 BP1, which were significantly prohibited in GSTT1 cells (Figure 3D). These data indicated that DNA damage is constitutively activated in response to PAH and could be protected by GSTT1 expression.

GSTT1 Inhibits PAH-mediated Lymphoma Cell Proliferation

Upon treatment with normal saline, there is no difference of cell growth between the FU and GSTT1 groups. However, cell growth was enhanced in Hydroquinone-treated FU cells, which could be significantly reduced by ectopic expression of GSTT1 (Namalwa, P = 0.045 and Jurkat, P = 0.043, respectively, Figure 4A). Cell proliferation was further determined by EdU assay. Comparing with the FU groups, EdU-positive cells in the GSTT1 groups were accordingly reduced (Namalwa, P = 0.043 and Jurkat, P = 0.039, respectively, Figure 4B). Cell cycle analysis showed a lower percentage of S-phase cells in the GSTT1 groups than in the FU groups (Namalwa, 17.5±0.52% vs 14.3±0.55%, P = 0.013, and Jurkat, 14.1±0.44% vs 11.8±0.54%, P = 0.032, respectively). As for cell apoptosis, neither Namalwa nor Jurkat cells showed obvious change in the percentage of ANX-V-positive cells between the FU and GSTT1 groups when treated with Hydroquinone (Figure 4C).

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Figure 4. GSTT1 expression inhibits PAH-mediated lymphoma cell proliferation.

A: Effect of GSTT1 expression on cell proliferation. 3×10⁵ cells treated with normal saline or Hydroquinone were seeded and cell number was counted at 48 h by typan blue. Data represent Mean±S.E. of densitometric values from three individual experiments. B: EdU assay of Namalwa and Jurkat cells treated with normal saline or Hydroquinone. C: Cell apoptosis analyzed by flow cytometry. Histography indicates Mean±S.E. from three individual experiments. D: Key proteins of DNA damage and cell cycle detected by Western blot in Namalwa and Jurkat cells with or without Hydroquione treatment. *P<0.05 comparing with the FU cells.

https://doi.org/10.1371/journal.pone.0089302.g004

By Western blot, expressions of cell cycle regulation proteins were assessed with or without Hydroquinone treatment. In Hydroquinone-treated Namalwa and Jurkat cells, GSTT1 expression was consistent with downregulation of MYC, pCHK1 and nuclear 53 BP1, while pCHK2, total CHK1 and CHK2 levels remained constant (Figure 4D).

GSTT1 Retards Tumor Formation of PAH-treated Lymphoma Cells

To assess the carcinogenic potential in vivo, the FU and GSTT1 cells were treated with Hydroquinone as mentioned above and injected subcutaneously in nude mice (Figure 5A). Expression of GSTT1 could significantly prolong the latency of tumor formation. Of note, 2×10⁶ Namalwa cells derived from the FU group could form tumors in 10 of 12 nude mice in 4 weeks. However, when 2×10⁶ cells from GSTT1 group were injected, only 4 tumors were found in 12 nude mice (P = 0.013). Similar results were obtained in Jurkat cells: tumor were formed in all mice at 4 weeks injected with 1×10⁷ FU cells, but only 6 of 12 mice in those treated with the same amount of GSTT1 cells (P = 0.014).

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Figure 5. GSTT1 expression reduces tumorogeneity of PAH-treated lymphoma cells.

A: Tumor formation in nude mice. Indicated amount of Hydroquinone treated cells were injected subcutaneously and tumorigenicity was reported as numbers of tumors formed per numbers of mice injected. B: Expression of GSTT1, Ki67 and MYC detected in tumor tissues by immunohistochemistry assay, as well as pCHK1, γH2AX and 53 BP1 by immunofluorescence assay. *P<0.05 comparing with the FU cells.

https://doi.org/10.1371/journal.pone.0089302.g005

To search for in situ evidence of DNA damage and tumor cell proliferation, immunoflurescence assay of γH2AX, 53 BP1 and pCHK1, as well as immunohistochemistry assay of Ki67 and MYC were performed on mice tumor sections. In parallel with in vitro results, all markers were increased in the FU groups, compared with the GSTT1 groups following Hydroquinone treatment (Figure 5B).

GSTT1 Knock-down Stimulates Lymphocyte Proliferation in PAH-treated Zebrafish

After searching the zebrafish genome data base (Zv9), two GSTT1 genes were identified, referred as gstt1a and gstt1b, sharing part of nucleotide acids identical to human GSTT1 gene. To confirm the evolutionary conservation of GSTT1, we investigated the distribution of genes located adjacent to the GSTT1 locus in zebrafish and in human genomes. As shown in Figure S1A, 9 genes, along with GSTT1, define an approximately 840-kb genomic region on human chromosome 22, which is syntenic to the zebrafish gstt1a genomic locus on linkage group 8 and zebrafish gstt1b on linkage group 21. Thus, zebrafish gstt1a and gstt1b are evolutionarily conserved orthologs of human GSTT1.

Then the temporal and spatial expression patterns of gstt1a and gstt1b were examined in zebrafish embryos from 0.25 h to 120 hpf by WISH using digoxigenin-labeled antisense RNA probe. Strong signals were detected in the blastomeres of the two-cell stage and the shield period (6 h, Figure S1B), indicating that both gstt1a and gstt1b transcripts were maternally derived and played a role in early embryonic development. Although both genes exhibited similar patterns of expression during the early stage, differential expression was observed after 30 hpf: high levels of gstt1a transcripts were mostly restricted to the liver (2d), while gstt1b transcripts gradually disappeared after 30 hpf and was no longer observed after 3d (Figure S1B).

To further determine the function of GSTT1 on normal lymphocytes, morpholino was applied to efficiently block gstt1a and gstt1b gene expression in zebrafish. Injection of MO of gstt1a at the dosage of 8 ng per embryo, but not mismatch oligo control, was able to completely suppress the EGFP expression of co-injected capped gstt1a-EGFP mRNAs reporter (Figure S1C, left panels), indicating that the antisense RNA that could effectively silence gene expression. Similar results were obtained in gstt1b (Figure S1C, right panels).

Exposure of BaP, a carcinogenic PAH, was conducted at the concentration of 10 µg/L, based on previous experiments investigating the teratogenicity of PAH on zebrafish [14], [15]. Zebrafish embryos were microinjected with gstt1a and gstt1b morpholinos or 5 bp mismatch oligo controls, and treated with BaP from 24 hpf to 4 d. DMSO was used as the solvent control at a final concentration of 0.1%. At least 100 embryos were included in each group.

Comparing with those injected with mismatch oligos, gstt1a and gstt1b silenced embryos showed an increase of the rag1 signal in the thymus under exposure to BaP, as detected by WISH (Figure 6A, right panels). No significant difference of the rag1 signal was observed in embryos treated with DMSO (Figure 6A, left panels).

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Figure 6. Knock-down of gstt1a and gstt1b promotes lymphocyte proliferation exposed to BaP.

A; WISH images showed the rag1 expression in the thymus (arrows) of differently treated 5 dpf embryos. B: In situ analysis of myca at 5 dpf. The morphants showed increased expression of myca in microinjected gstt1a and gstt1b morpholino exposed to BaP (Left panels), semi-quantitative PCR showed similar expression pattern in embyos (Right panels). C: Ultrastructure of thymic lymphocytes from 5 dpf larvae exposed to BaP. Images represent results from three independent experiments and each group contains 30 morphants.

https://doi.org/10.1371/journal.pone.0089302.g006

To confirm that MYC activation is also involved in PAH-associated stimulation of lymphocyte proliferation in zebrafsh, the expression of myca, zebrafish homologue of human MYC, was assessed in BaP-treated embryos at 5 d. In accordance with rag1 expression, myca expression was significantly higher in embryo injected with gstt1a and gstt1b morpholinos than those injected with mismatched oligos by WISH (Figure 6B, left panels) and by semi-quantitative PCR (Figure 6B, right panels).

The structure of the thymus in 5 d-embryos was examined by transmission electron microscopy (Figure 6C). Ultrastructural analysis of both cells (microinjected with gstt1a and gstt1b morpholino or oligo control) revealed no sign of apoptosis. At a higher magnification, abundant euchromatin and robust mitochondrias were observed in BaP-treat gstt1-silenced lymphocytes, possibly related to a hyper-proliferative status of the cells.

Therefore, in addition to lymphoma cells, inactivation of GSTT1 was able to confer a proliferative advantage induced by PAH in their normal counterparts.