Parental benzo[a]pyrene exposure impacts histone modifications in osteoblast subpopulations

Abstract

Environmental stressors, such as benzo[a]pyrene (BaP), have been repeatedly associated with developmental bone defects in offspring after parental exposures. Chemical modifications along the histone 3 protein (H3) and histone 4 protein (H4) tails are crucial for osteoblast differentiation. Therefore, H3K4me3, H3K9me3, H3K27me3, H3K27ac, and H4K5ac/K8ac/K12ac have been assessed by immunofluorescence. F1 adults from a transgenic twist:dsred/col10a1:gfp medaka (Oryzias latipes) strain with/without parental BaP exposure were assessed to yield novel data on the histone code of osteoblasts and allow quantification of parental environmental pollutant exposure’s interference with chromatin structure regulation. In twist⁺ cells, BaP exposure significantly reduced H3K9me3 marks in both male and female fish. Significant reductions of H3K9me3 and H4K5ac/K8ac/K12ac were observed in col10a1⁺ cells of male fish with parental BaP exposure. Notable sex-specific differences existed across histone modifications in these osteoblast subpopulations. Understanding the relationship between histone modifications and bone health will improve the assessment of ecological risk and public health impact of BaP pollution and further support the hypothesis that BaP-induced histone modifications are inherited over generations and involved in bone formation in an osteoblast subpopulation-specific manner.

Introduction

Histone post-translational modifications (PTMs) are central to chromatin remodelling and gene regulation. Ancestral exposure of medaka (Oryzias latipes) to the polycyclic aromatic hydrocarbon benzo[a]pyrene (BaP) is hypothesized to perturb this system: offspring of BaP-exposed parents display altered bone mineralization and dysregulated expression of histone-modifying enzymes [1,2]. Prior work documented BaP-induced changes in microRNA profiles and locus-specific DNA methylation [1,3], histone PTMs themselves have not been examined. BaP exposure can induce mutagenicity, genotoxicity, carcinogenicity, teratogenicity, endocrine disruption, immunotoxicity, neurotoxicity, cardiotoxicity, and osteotoxicity in vertebrates [4]. Concentrations vary from 0.1 ng/L to the 8.6 µg/L in polluted locations [5–7].

Specific histone marks tightly control osteogenesis. Loss of the activating trimethyl mark on histone 3 lysine 4 (H3K4me3) at the runt-related transcription factor 2 (Runx2) and osterix promoters is linked to osteoporotic bone in mice [8–10]. Trimethylation of histone 3 lysine 9 (H3K9me3) compacts heterochromatin, silencing non-osteogenic genes and steering mesenchymal stem cells (MSCs) towards the osteoblast lineage [11–13]. Active acetyl marks also shape skeletal fate: histone 3 lysine 27 acetylation (H3K27ac) supports balanced bone formation and resorption [14,15], while histone 4 lysine 5 acetylation (H4K5ac) opens chromatin to facilitate MSC commitment to osteoblasts [16,17]. Histone 4 lysine 8 acetylation (H4K8ac) promotes osteoclastogenic wingless-related integration site 5a (Wnt5a) transcription and biases MSCs towards adipogenesis at the expense of osteoblasts [18–20]. Age-related gain of histone 4 lysine 12 acetylation (H4K12ac) reduces Runx2 promoter activity, dampening osteogenic differentiation [21].

We hypothesize that parental BaP exposure reprogrammes the histone code of discrete osteoblast subpopulations in a sex-specific manner. Sexual dimorphism in endocrine signalling can modulate the epigenetic machinery [22]. Whole-genome surveys reveal sex-dependent chromatin states in bone tissue [23]. To resolve cell-type-specific PTMs, we employ the twist:dsRed/col10a1:gfp transgenic medaka line. Twist marks early osteochondral precursors residing near vertebral centra and directly interacts with Runx2 [24,25]. Collagen 10a1 (col10a1) labels osteochondral progenitors and premature osteoblasts [26].

Results

Parental BaP exposures in general affected bone cell subpopulations. Col10a1⁺ cell coverage per bone area was significantly reduced in the male BaP group compared to the male control group (P  <.05; Fig. 1).

Figure 1.

(A) Average percentage of fluorescent area per bone area for twist⁺ and col10a1⁺ cells across sex and treatment groups: female control (cyan), female BaP (blue), male control (green), and male BaP (dark green). Comparison between male control and male BaP in col10a1⁺ cells (*P < .05). Bars depict means ± SEM. (B) Transgenic (twist:dsred/col10a1:gfp) parentally BaP-exposed F1 adult medaka posterior vertebrae regions at 10× magnification. From left to right: (1) control female, (2) BaP female, (3) control male, and (4) BaP male. Top to bottom: (A) brightfield, (B) twist⁺, and (C) col10a1⁺; IS = intervertebral segment; c = centrum; vs = vertebral segment; scale bars indicate 50 µm.

Histone modifications differed with osteoblast maturation (Fig. 2). H3K9me3, H3K27me3, H3K27ac, and H4K5ac/8ac/12ac significantly increased from male twist⁺ cells to male col10a1⁺ cells (Fig. 2). H3K27me3 and H4K5ac/8ac/12ac were found significantly increased in male col10a1⁺ cells compared to female col10a1⁺ cells.

Figure 2.

Average cellular fluorescence ratio per bone area for twist⁺ and col10a1⁺ cells in female and male F1 adult medaka vertebrae across histone modifications: H3K4me3, H3K9me3, H3K27me3, H3K27ac, and H4K5ac/K8ac/K12ac. Bars represent mean ± SEM. Significant differences between groups are indicated by asterisks (*P < .05, **P < .01, ***P < .001) based on one-way ANOVA followed by Tukey’s post hoc test; n = 10.

Parental Bap exposure changed male and female histone modification in twist⁺ cells (Fig. 3). H3K9me3 was significantly reduced in twist⁺ osteoblast progenitors of both male (P <.01) and female (P <.05) parentally BaP-exposed individuals. H4K5ac/K8ac/K12ac expression in twist⁺ cells was significantly higher in the male control group than in the male BaP group (P <.001). Evaluating sex differences, the female control showed significantly less fluorescence for H4K5ac/K8ac/K12ac in twist⁺ cells than the male control (P <.05). Neither the measurements of the generic H3 antibody nor the negative control revealed significant differences.

Figure 3.

(A) Boxplots depict the area of overlap (µm²) between transgenic twist⁺ fluorescence and the histone modifications antibody fluorescence: H3K4me3, H3K9me3, H3K27me3, H3K27ac, H4K5ac/K8ac/K12ac, H3Pan, and a negative control. The four groups shown represent female control (cyan), female BaP-treated (light blue), male control (green), and male BaP-treated (dark green). Asterisks indicate statistically significant differences between control and treatment (*P < .05, **P < .01, ***P < .001); # indicates statistically significant differences between sexes (P < .05). (B) Immunofluorescence overlap of H3K9me3 and osteoblast subpopulations on parentally BaP-exposed and non-exposed F1 adult medaka. Control: H3K9me3-twist control male; BaP: H3K9me3-twist parentally BaP-exposed male; scale bar = 50 µm.

Parental Bap exposure impacted histone modifications in col10a1⁺ cells from male and female bone tissue (Fig. 4). The male parentally BaP-exposed group had significantly reduced H3K9me3 fluorescence in col10a1⁺ cells compared to the male control (P <.001). Male control samples showed a significantly higher H3K9me3 antibody fluorescence of col10a1⁺ cells than the female control (P <.05), highlighting sex differences. H4K5ac/K8ac/K12ac was significant decreased in col10a1⁺ cells from the male BaP group compared to the male control group (P <.001). Sex differences were observed for H4K5ac/K8ac/K12ac, and the male control had significantly increased antibody fluorescence in col10⁺ cells than the female control group (P <.001).

Figure 4.

(A) Fluorescent area overlaps between col10a1⁺ cells and various histone modification antibodies in medaka vertebrae, separated by sex and treatment groups (BaP and control). Boxplots represent the quantified overlap (µm²) between col10a1 fluorescence and the histone modifications H3K4me3, H3K9me3, H3K27me3, H3K27ac, H4K5ac/K8ac/K12ac, H3Pan, and a negative control. Statistical analysis was performed using ANOVA followed by Tukey’s post hoc tests where applicable. # indicates statistically significant differences between sexes and *** indicates P < .001, between control and parentally exposed individuals. (B) Immunofluorescence overlap of H3K9me3 and osteoblast subpopulations on parentally BaP-exposed and non-exposed F1 adult medaka. Control: H3K9me3-twist control male; BaP: H3K9me3-twist parentally BaP-exposed male; scale bar = 50 µm.

Discussion

Parental BaP exposure impacts histone modification profiles in medaka vertebrae, affecting the histone marks H3K9me3 and H4K5ac/K8ac/K12ac in twist⁺ and col10a1⁺ osteoblast subpopulations. Mapping these modifications within discrete osteoblast subpopulations adds a novel mechanistic layer to how contaminants reshape vertebrate skeletal development.

Higher H3K27me3 and H4 acetylation in col10a1⁺ cells from male control fish revealed subtle sex-specific chromatin organization. H3K27me3 regulates BMP-2 expression and osteogenesis, and its demethylation can be driven by oestrogen receptor-α activation, explaining male–female contrasts [27,28]. H4K5ac/K8ac/K12ac facilitate transcription of osteogenic genes (alkaline phosphatase, osteocalcin, Runx2) [18, 21, 29], hinting that endogenous hormone signalling imprints sex-biased chromatin states [30–32].

Changes in histone modifications were identified in twist⁺ mesenchymal-derived stem cells vs. col10⁺ osteoblast progenitors and more prominent in male vs. female individuals, indicating the importance of histone modifications on cell differentiation, including osteoblasts. With differentiation into osteoblast progenitors, H3K9me3, H3K27me3, H3k27ac, and H4k5ac/8ac/12ac marks were found increased, possibly reflecting the complexity of gene expression regulation during cell lineage differentiation [33]. Concomitant upregulation of repressive and enhancer histone marks has been previously demonstrated for osteoblast development [34]. The increase in H3 and H4 acetylation marks the transition from a closed state towards a transcriptionally active chromatin to enable expression of bone-related genes and transcription factors [29, 33, 35]. H3K9me3 and H3K27me3 are promoters of osteogenic differentiation [36].

This study elucidated that parental BaP exposure may alter key histone modifications in osteoblast subpopulations in the Japanese medaka, specifically in twist⁺ mesenchymal-derived stem cells and col10⁺ osteoblast progenitors, which play sequential roles in osteoblast differentiation. The histone modifications explored in the osteoblast cell types (twist⁺ and col10a1⁺) highlight a potential mechanism through which parental BaP exposure may disrupt the osteoblast function and bone development in a specific sex-dependent manner.

BaP exposure significantly reduced H3K9me3 marks in both male and female fish twist⁺ cells. H3K9me3 is a repressive histone modification important for heterochromatin formation and transcriptional regulation, maintaining cell differentiation [14]. This modification is known to lead to osteogenic differentiation by activating the canonical WNT signalling pathway, essential for osteoblast lineage commitment [37]. The reduction of H3K9me3 in twist⁺ cells suggests that BaP disrupts early osteoblast differentiation, potentially compromising the ability of these cells to fulfil normal developmental pathways. The loss of H3K9me3 in twist⁺ cells may also lead to reduced heterochromatin formation, resulting in genome instability and transcriptional deregulation [38]. The alterations in histone profiles could interfere with twist regulatory function in MSC differentiation, impairing osteoblast development at its premature and early stages. The significant decrease of H4K5ac/K8ac/K12ac in twist⁺ cells suggests a compromised chromatin accessibility that could further disrupt the transcriptional needs required for early osteoblast differentiation. H4K5ac is critical for MSC differentiation [39], and its reduction in BaP-exposed males likely shows an early impairment in osteoblast precursor cells, ultimately affecting later stages of bone development. As twist⁺ cells transition into col10a1⁺ expressing cells during osteoblast differentiation, BaP exposure continues to affect histone modifications, particularly in male Japanese medaka.

In col10a1⁺ cells, male BaP-treated fish showed significantly reduced H3K9me3 and H4K5ac/K8ac/K12ac. H3K9me3 maintains transcriptional repression and WNT pathway activation necessary for bone formation [13]. The reduction of H3K9me3 in col10a1⁺ cells may disrupt osteoblast maturation, potentially compromising bone matrix production and mineralization in BaP-exposed offspring, as evidenced in [1]. The decrease in H4K5ac/K8ac/K12ac marks in col10a1⁺ cells further showed BaP’s impact on chromatin structure and gene expression in bone-forming cells, with inhibition of H4K5ac impacting MSC commitment to osteoblasts [16,17] and reduced H4K8ac and H4K12ac marks possibly indicate a compensatory mechanism [18–21].

Lower H3K9me3 may facilitate access of transcriptional machinery to gene promoters enabling gene expression, while H4 lysine acetylation is indicative of active transcription. A reduction of H3K9me3 may impact differentiation towards the osteoblastic lineage through Runx2 deregulation [11–13, 40, 41], an osteoblast differentiation promotor during early development, but a differentiation repressor at later osteoblast maturation [42–45]. Reduced H4K5ac, H4K8ac, and H4K12ac can lead to decreased expression of osteoblast-specific genes (osteocalcin, bone sialoprotein), and support Runx2 promotor activity [21, 41]. The BaP-induced reduction of col10a1⁺ cells and the reduction of H3K9me3 and H4 acetylation hint towards the repression of later osteoblast differentiation [1] mediated by increased Runx2 transcription [46]. The histone modification dysregulation in col10a1⁺ cells, following similar changes in twist⁺ cells, suggests that BaP exposure impairs osteoblast differentiation at multiple stages, with ongoing effects on bone development. The impact of BaP exposure on histone profiles was more pronounced in male Japanese medaka, suggesting a sex-specific sensitivity to environmental pollutants like BaP within osteoblast subpopulations, which is in line with the phenotype observed at the tissue level [1]. Ancestral BaP exposure in Japanese medaka, specifically in males, led to bone thinning and altered bone integrity within the F3 generation in conjunction with deregulated mRNA and miRNA expression, affecting osteoblast differentiation and cellular changes [47]. The observed reduction in bone mineralization in BaP-exposed males may be a direct consequence of disrupted histone modifications in both twist⁺ and col10a1⁺ cells, changing osteoblast differentiation and bone formation at multiple stages.

These findings strengthen evidence that parental BaP exposure epigenetically reprogrammes bone cells. Future work should pinpoint BaP-sensitive osteogenic pathways and apply high-resolution chromatin assays in defined osteoblast subpopulations to clarify transgenerational effects on bone formation.

Materials and methods

Model organism

Japanese medaka (Oryzias latipes) is an established teleost model whose osteogenic genes and regulatory circuits are highly conserved with mammals [48,49]. We used the double-transgenic twist:dsRed/col10a1:nlGFP line, kindly provided by C. Winkler (National University of Singapore) and maintained for several generations at Texas A&M University-Corpus Christi (TAMU-CC) [44]. All procedures were approved by the TAMU-CC IACUC (#2023-0002).

Benzo[a]pyrene exposure

Four-month-old F0 transgenic fish were exposed to 1 µg/L benzo[a]pyrene (BaP) for 21 days; controls received 0.0005% dimethylsulfoxide (DMSO)/ethanol (1:4) carrier [47]. This BaP exposure regime is environmentally relevant and induced transgenerational bone toxicity [1,47]. Five replicate 20 L tanks each contained 10 breeding pairs. Seventy-five per cent water changes were performed every second day before BaP administration. On days 22–23, three additional water exchanges eliminated residual BaP before eggs were collected daily. F1 embryos were reared under standard conditions [50] and grown to 6 months.

Tissue fixation and Cryo-sectioning

Medaka tissues (n = 5 per sex and treatment) were dissected to isolate vertebral columns. Samples were embedded in fresh optimal cutting temperature compound (OCT), snap-frozen on dry ice, foil-wrapped, and stored at –80°C. Blocks equilibrated in a cryostat (–15°C) for 30 min were sectioned at 25 µm. Slides were stored in ideal preservation conditions (−20°C) for the immunohistochemistry/immunofluorescence (IHC/IF) staining.

Immunostaining

Slides were rehydrated in phosphate buffered saline (PBS). Non-specific binding was blocked for 30 min in 1% BSA/0.3% Triton X-100 in PBS. Sections were incubated overnight (2–8°C) with rabbit monoclonal antibodies against H3Pan (Cat# C15200011), H3K4me3 (Cat# C15410003) [51, 52], H3K9me3 (Cat# C15410193) [51], H3K27me3 (Cat# C15410069) [51, 53], H3K27ac (Cat# C15410196) [52], and H4K5/8/12ac (against the region of histone H4 containing the acetylated lysines 5, 8, and 12; Cat# C15410021) (Hologic Diagenode; 1:500). All antibodies showed universal species reactivity and have been tested by the supplier for target specificity through cross-reactivity testing with over 90% [54]. After two PBS washes, Alexa Fluor™ 405 goat anti-rabbit IgG (Thermo Fisher; 10 µg/ml) was applied for 60 min, followed by three washes. Slides were mounted in FluoroShield™. Images were obtained with an Olympus BX-53. Representative images for H3K27me3 taken with a Nikon AX NSPARC are provided in Fig. 5.

Figure 5.

Representative images with confocal microscopy (Nikon AX/AX R with NSPARC) (A) brightfield adult male bone section, (B) adult male bone section with col10:gfp signal, (C) adult male bone section with twist:rfp signal, (D) adult male bone section with H3K27me3 signal, and (E) higher resolution of H3K27me3 signal.

Data analysis

Investigators were blinded for treatment. Regions of interest (vertebral bone) were delineated manually in FIJI/ImageJ [55]. Images were converted to 8-bit and analysis was implemented in Jupyter Notebook [56] with Python 3 scripts [57]. Bone area and fluorescence intensities were quantified via the pyimagej interface. Histone-signal overlays with twist⁺ or col10a1⁺ masks generated lineage-specific PTM readouts.

Data were analysed in R 4.4.1 [58]. Normality and equality of variances were assessed with Shapiro–Wilk’s test and Levene’s test. Histone-specific effects, sex effects, and their interaction were tested with one- or two-way ANOVA followed by Tukey’s honestly significant differences (HSD) post hoc comparisons (α = 0.05). Data are presented as mean ± SEM and adjusted P values (P < .05) indicated statistical significance.

The average fluorescent area indicates the proportion of each vertebral segment occupied by the transgenic fluorescence marker, reflecting differences in osteoblast subpopulation coverage. The average cellular fluorescence ratio represents the percentage overlap between the transgenic marker and histone antibody signals, assessing subpopulation-specific histone modification changes. The fluorescent area overlap measures the (µm) region where both signals coincide, allowing evaluation of histone modifications independent of cell subpopulation size.

Author contributions

Alexis S. Trujillo (Data curation, Formal analysis, Investigation, Visualization, Writing — original draft), Remi O. Labeille (Methodology, Software, Formal analysis), Rijith Jayarajan (Data curation), Dylan Mack (Methodology, Formal analysis), and Frauke Seemann (Conceptualization, Funding acquisition, Methodology, Project administration, Resources, Supervision, Validation, Writing — review & editing)

Conflict of interest

The authors have no relevant financial or non-financial interests to disclose.

Ethics declarations

This research did not involve human participants, their data, or biological material. All animal work was approved by the Texas A&M University-Corpus Christi Institutional Animal Care and Use Committee (TAMU-CC-IACUC-2023-0002) and adhered to the ARRIVE guidelines.

Data availability

The data and fish lines used in this study are available upon request to the corresponding author. The analysis pipeline is available at Rlabeille/Parental-Benzo-a-pyrene-exposure-impacts-histone-modifications-in-osteoblast-subpopulations