key: cord-0984893-gf9hxrj7
authors: Larsen, Michele Campaigne; Almeldin, Ahmed; Tong, Tiegang; Rondelli, Catherine M.; Maguire, Meghan; Jaskula-Sztul, Renata; Jefcoate, Colin R.
title: Cytochrome P4501B1 in bone marrow is co-expressed with key markers of mesenchymal stem cells. BMS2 cell line models PAH disruption of bone marrow niche development functions
date: 2020-06-14
journal: Toxicol Appl Pharmacol
DOI: 10.1016/j.taap.2020.115111
sha: 2b8faec4658ed847c3e85a89620b24193046017a
doc_id: 984893
cord_uid: gf9hxrj7

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous pollutants that are metabolized to carcinogenic dihydrodiol epoxides (PAHDE) by cytochrome P450 1B1 (CYP1B1). This metabolism occurs in bone marrow (BM) mesenchymal stem cells (MSC), which sustain hematopoietic stem and progenitor cells (HSPC). In BM, CYP1B1-mediated metabolism of 7, 12-dimethylbenz[a]anthracene (DMBA) suppresses HSPC colony formation within 6 h, whereas benzo(a)pyrene (BP) generates protective cytokines. MSC, enriched from adherent BM cells, yielded the bone marrow stromal, BMS2, cell line. These cells express elevated basal CYP1B1 that scarcely responds to Ah receptor (AhR) inducers. BMS2 cells exhibit extensive transcriptome overlap with leptin receptor positive mesenchymal stem cells (Lepr+ MSC) that control the hematopoietic niche. The overlap includes CYP1B1 and the expression of HSPC regulatory factors (Ebf3, Cxcl12, Kitl, Csf1 and Gas6). MSC are large, adherent fibroblasts that sequester small HSPC and macrophage in the BM niche (Graphic abstract). High basal CYP1B1 expression in BMS2 cells derives from interactions between the Ah-receptor enhancer and proximal promoter SP1 complexes, boosted by autocrine signaling. PAH effects on BMS2 cells model Lepr+MSC niche activity. CYP1B1 metabolizes DMBA to PAHDE, producing p53-mediated mRNA increases, long after the in vivo HSPC suppression. Faster, direct p53 effects, favored by stem cells, remain possible PAHDE targets. However, HSPC regulatory factors remained unresponsive. BP is less toxic in BMS2 cells, but, in BM, CYP1A1 metabolism stimulates macrophage cytokines (Il1b > Tnfa> Ifng) within 6 h. Although absent from BMS2 and Lepr+MSC, their receptors are highly expressed. The impact of this cytokine signaling in MSC remains to be determined.

Polycyclic aromatic hydrocarbons (PAHs) are major health risk factors through the association of smoking with lung cancer and their contributions to multiple adverse effects of vehicle air particulates (Bostrom, et al. 2002; Castano-Vinyals, et al. 2004; Layshock, et al. 2010; Moorthy, et al. 2015; Yang, et al. 2019) . In present times, we can also add the impact of smoking and environmental combustion pollutants on health outcomes from COVID-19 infection (Li Volti, et al. 2020) . The metabolism of these chemicals causes tissue injury and carcinogenic mutations (Bolton and Dunlap 2017; Bostrom, et al. 2002; Castano-Vinyals, et al. 2004; Moorthy, et al. 2015; Yang, et al. 2019) , but also impacts the immune system (O'Driscoll, et al. 2018) , notably from effects in the bone marrow (BM) (Larsen, et al. 2016; N'Jai A, et al. 2011; N'Jai A, et al. 2010) .

In previous work, we have shown that hematopoietic stem and progenitor cells (HSPC) in mouse BM respond with remarkable speed and selectivity to PAHs (Larsen, et al. 2016) .

These effects have wide systemic consequences, notably in the spleen and thymus (Larsen, et al. 2016) . This disruption, which is mediated by cytochrome P450 1B1 (CYP1B1), completes within a few hours, but PAHs can also generate a rapid protection process. The PAH selectivity of these opposing processes is dependent on specific metabolites. Here, we P450 in BMS2 cells.

HSPC differentiate into lymphoid, myeloid, and erythroid lineages (Lai and Kondo 2006) . that migrate to sites of injury where they generate inflammatory and repair responses (Li and Ikehara 2013) . MS C provide essential support for HSPC differentiation by releasing specific support cytokines, including Cxcl12, Csf and Ilf7 (Crane, et al. 2017) . Subsets of MSC, notably leptin receptor positive (Lepr+MSC) cells, undergo self-renewal, directed by Cxcl12 and Kitl/Scf (Galan-Diez and Kousteni 2018) . BMS2 cells lack Lepr expression and the capacity for self-renewal, but effectively support lymphoid progenitors (Rondelli, et al. 2016) . Here, we show that many of the most abundant genes in Lepr+MSC are also highly expressed in BMS2 cells , including CYP1B1. This led us to hypothesize that PAH metabolism in Lepr+MSC causes the rapid and extensive suppression of HSPC expansion in BM. In this respect, BMS2 cells should provide an informative model for PAH effects in the vascular hematopoietic niche.

BMS2 cells are used in these studies to address the remarkable opposing effects of PAH on BM lymphoid and myeloid progenitor cells (N'Jai A, et al. 2011) . The adverse effects are realized predominantly through local CYP1B1-mediated bioactivation to PAHDE, rather than by poorly expressed cytochrome P450 1A1 (CYP1A1) (Heidel, et al. 1998) . 7, 12dimethylbenz[a]anthracene (DMBA) extensively suppresses specific colony forming activities (CFU) within 6h, through a process that is CYP1B1-dependent (N'Jai A, et al. 2010) . The number of colonies quantifies the proportion of initial active progenitors (lymphoid, myeloid or erythroid), while the colony size indicates the rate of expansion. Flow cytometry analyses show matching DMBA effects on HSPC (Larsen, et al. 2016) . A key feature of the PAH suppression response is a complete insensitivity to induction by PAHs. This matches the novel regulation of CYP1B1 in BMS2 cells, which is likely to extend to Lepr+MSC. Presumably, CYP1B1 has an important, but Diez and Kousteni 2018) . BMS2 cells, and their primary counterparts, reproduce the substantial CYP1B1 basal expression, without stimulation by AhR activators (Heidel, et al. 1998) . Importantly, AhR deletion shows that much of the basal expression is lost in AhR-null MEFs (Alexander, et al. 1997) and BM MSC (Heidel, et al. 1998) . This pattern of regulation is also observed in OP9 MSC, while basal expression in MEFs is substantially enhanced by AhR activation (Rondelli, et al. 2016) .

This basal CYP1B1 expression in BMS2 cells is regulated via dual SP1 complexes in the proximal promoter (Wo, et al. 1997) , in partnership with complexes in the Ah enhancer region (AhER) and other upstream enhancers (Zhang, et al. 1998; Zhang, et al. 2003) . Constitutive CYP1B1 is also activated by AhR in absence of an exogenous ligand, via disruption of cell adhesion (Cho, et al. 2004; Ziegler, et al. 2016) and by endogenous ligands formed from tryptophan (Seok, et al. 2018; Villa, et al. 2017) . This work shows that CYP1B1 expression in MSC is highly susceptible to autocrine regulation. CYP1B1 metabolizes DMBA and BP to PAHDE, which generate DNA adducts and DSB that activate ATM kinase to phosphorylate p53 (Ganesan, et al. 2013; Gao, et al. 2008) . p53 activation plays an essential role in both HSPC suppression (Page, et al. 2003; Teague, et al. 2010 ) and stabilization of the MSC-HSPC niche (Phinney and Prockop 2007) . We show, here, that the BMS2 transcriptome exhibits expression asymmetry in the pairing of receptor and their respective activators. We identify several strongly expressed pairs that could potentially affect autocrine regulation, notably Pdgfa and Pdgfrb. By contrast, MSC macrophage cytokines (Ifng, Il1b and Tnf) are absent, but their respective receptors are strongly expressed. This BMS2 model for MSC fits an emerging picture of the hematopoietic niche, driven by signaling from macrophage (Chow, et al. 2011) , with bidirectional effects on HSPC (Schajnovitz, et al. 2011) .

To better understand the in vivo MSC responses to DMBA and BP, we examined their effects on gene expression in BMS2 cells. We resolve direct AhR effects produced within 8h and metabolite-driven responses that only appear after 8h. This delay accommodates the multistep generation of PAHDE. The participation of the ATM/p53 is evident from the selectivity of gene responses, which are heavily weighted to DMBA. There is only a modest preference seen for general p53 activation.

Single cell sequencing of eluted BM cells shows multiple clusters, based on statistical analyses of mRNA abundance (Lai and Kondo 2006) . Hematopoietic progenitors are retained in the adherent fraction through surface attachment to the far larger, adherent fibroblastic MSC, which contribute only about 5 percent of the mRNA (Hu, et al. 2018) . Here, we compare the pattern of highly expressed mRNA in BMS2 cells with the corresponding mRNA in the adherent BM fraction. A set of functional MSC factors are identified in adherent BM cells in the range of (4 +/-3 percent). We also identify factors that are expressed in BMS2 cells but have relatively much lower expression in the MSC of adherent BM cells. We identify a twelve-gene functional core that shares expression in BMS2 cells, the adherent BM fraction and Lepr+MSC. Notably, their expression is highly correlated with CYP1B1, but not affected by DMBA or BP. CYP1A1 or CYP1B1 (V79-hCYP1A1 and V79-hCYP1B1, respectively) were provided by Dr. J.

Doehmer (Luch, et al. 1998; Luch, et al. 1999) . All cells were cultured under standard conditions (37°C, 5% CO 2 in saturated atmospheric humidity) in FBS-supplemented media (BMS2, RPMI 1640; C3H10T1/2, DMEM; V79, DMEM high glucose supplemented with pyruvate and G418) (Fisher Scientific, Waltham, MA). Physically separated co-cultures, which shared media, were used for the analyses of secreted factor exchange. The cell lines lose CYP1B1 expression when near confluence and, therefore, all cultures were completed at an initial 70 percent of confluence, unless otherwise stated. Adipogenic differentiation in primary BM cells and the BMS2 cell line was completed as previously described (Jefcoate, et al. 2008) . Colony forming unit (CFU) assays were completed using kits purchased from Stem Cell Technologies, according to manufacturers' protocol and as previously described (Larsen, et al. 2016; Rondelli, et al. 2016) .

Microarray analyses were completed in triplicate cultures of BMS2 cells treated with either DMBA, BP, or TCDD. Control cells were treated with vehicle (DMSO). RNA was isolated using Qiagen's RNeasy mini-kit (Hilden, Germany) and quantified using a Nanodrop spectrophotometer (Thermo Fisher Scientific; Waltham, MA). RNA integrity was assessed using denaturing formaldehyde-agarose gel electrophoresis. Cy3/5 labeling was completed using Agilent Technologies' Dual Color Gene Expression kit. Analyses were completed on the Whole Mouse Genome Microarray 4x44 slides, using the DNA Microarray Scanner and Feature Extraction Software (Santa Clara, CA). Cy5 values of greater than 50 were considered significantly above background for analysis. Expression is presented as fold change (Cy3/Cy5) from untreated cultures. Analysis was completed using the EDGE3 software package (Vollrath, et al. 2009 ). All p<0.01 were considered statistically significant.

Endogenous peroxides were quenched with 1% H 2 O 2 . Primary antibodies against phospho-p53 (Ser15) and phospho-H2AX (Ser139) were obtained from Cell Signaling (Beverly, MA) and used at a 1:500 dilution. Anti-mouse HRP-conjugated secondary antibody was purchased from Promega Corporation. Chemiluminescence was generated using a 1:1 reagent mixture of SuperSignal™ ELISA Femto Substrate (ThermoFisher Scientific) and normalized to DNA content using Hoecht florescence (Sigma-Aldrich, St. Louis, MO). Signal was quantified on a

BioTek Synergy 2 plate reader (BioTek, Winooski, VT).

SDS-PAGE (7.5%) immunoblot analyses were completed on total BMS2 and C3H10T1/2 cell lysates, as previously described (Cimafranca, et al. 2004) . Cells were treated with DMSO, TCDD or 3′-methoxy-4′-nitroflavone (MNF), as indicated, for 24h prior to isolation and lysate preparation. The CYP1B1 antibody was previously prepared in this laboratory (Savas, et al. 1997) . A non-specific background band serves as normalization for protein loading

Reporter constructs were previously prepared, as described (Zhang, et al. 2003) . These promoter constructs were transfected into BMS2 and C3H10T1/2 cells, at indicated cell densities, using electroporation at 200V. Promoter activity was measured with the Luciferase Reporter Assay System (Promega Corporation), as per manufacturer's instructions, using a Pharmingen Moonlight 3010 luminometer (BD Biosciences, San Jose, CA). MNF was used as an AhR antagonist, suppressing the AhER promoter activity in response to TCDD (10 nM, 24h) or DMSO solvent control. Data was expressed as a fold-induction of luminescence relative to untreated cells.

BMS2 and C3H10T1/2 cells were co-cultured to 80% of confluence in separate nested dishes, which provided by a barrier that allowed effective media exchange. To provide controls, each cell line was i n d i v i d u a l l y cultured in both compartments (two controls). The coculture and control cultures were maintained for 24h prior to recovery and electroporation at 200 V, as described for the standard promoter analyses. These electroporated cultures were separately stimulated with TCDD and DMSO solvent control for 24h. In an alternative procedure, either BMS2 cells or C3H10T1/2 cells were similarly transfected with the AhER reporter and co-cultured for 24h with an equal number of non-transfected cells, either of the same type or opposite type.

Data were graphed using GraphPad Prism software (version 8) and are represented as mean  SEM, n=3-6 observations/condition. CFU data from PAH-treated mice are expressed as the percent of the value of vehicle control (olive oil)-treated mice, with the control is set at 100 percent. Anova statistical analyses, followed by a Tukey post-test was completed unless otherwise stated. Student t-tests were completed using the unpaired, two-tailed constraints.

Agilent microarray data was analyzed by the EDGE3 software using the Limma analysis, which assesses significance based on ANOVA statistics (Vollrath, et al. 2009) 

In vivo treatment with DMBA has established that metabolism to reactive PAHDE by CYP1B1 J o u r n a l P r e -p r o o f results in 80 and 30 percent suppression of, respectively, lymphoid (preB) and myeloid (GM) CFU within 6h in WT mice ( Figure 1A) . BP is appreciably less active and all activities are lost in CYP1B1-ko mice ( Figure 1A ) (N'Jai A, et al. 2011) . Intraperitoneal (IP) (WT, Figure 1A ) and oral administration (all WT, Figure 1B ) of DMBA yielded similar 6h results, but analysis of serum PAH shows that elimination is complete within 24h after oral administration (N'Jai A, et al. 2010) , such that substantial restoration of preB and GM CFU occurs within 48h ( Figure 1B) , with near complete reversal after 168h (Larsen, et al. 2016) . This recovery is noteworthy because we find that DMBA-induced changes in transcription, mediated by CYP1B1 metabolism, are too slow to account for the acute CFU suppression, but closely parallels the recovery process (Table 1A) . These changes in CFU are paralleled in the T lymphocytes in the thymus, which also derive from BM progenitors, but with a delay of about 40h (Larsen, et al. 2016) . Flow cytometry analyses show parallel effects in HSPC progenitor populations (Larsen, et al. 2016) , which follow the CFU changes, preceding the mature BM populations. CYP1B1 and CYP1A1 convert PAHs, like DMBA and BP, to epoxides and phenols by monooxygenation reactions (Li, et al. 2017; Moorthy, et al. 2015) . Epoxides are unstable but are rapidly converted by microsomal epoxide hydrolase (Ephx1) to trans-dihydrodiols or spontaneously rearrange to phenols (Christou, et al. 1990; Gehly, et al. 1979; Pottenger and Jefcoate 1990) . In MSC, like BMS2 or C3H10T1/2, Ephx1 is present at about the same levels as CYP1B1 (Christou, et al. 1990; Savas, et al. 1994; Savas, et al. 1993) . The generation of PAHs to epoxides and then to dihydrodiols appears to be tightly coupled. In liver microsomes, supplemental Ephx1 greatly enhances dihydrodiol formation from CYP1A1, whereas in MSC the proportion of dihydrodiols is high and unaffected (Pottenger and Jefcoate 1990) . Dihydrodiol epoxides (PAHDE) are converted by a further CYP1B1 mono-oxygenation step that can be delayed until the starting PAH is sufficiently metabolized (Keller, et al. 1987) .

J o u r n a l P r e -p r o o f PAHDE DNA adducts in BM follow similar differences between DMBA and BP and dependence on CYP1B1 (Galvan, et al. 2005) . Comparison of BP and BP dihydrodiols in blood and BM, measured by HPLC analyses (N'Jai A, et al. 2011) , are consistent with origin from AhR-induced CYP1A1 in the liver (N'Jai A, et al. 2011; Uno, et al. 2004) . BM levels of BP, BP-dihydrodiol and BPDE adducts in WT and CYP1B1-ko mice establish that they are determined by intra-BM metabolism by CYP1B1 (Heidel, et al. 2000; N'Jai A, et al. 2011) .

In the BM, the functionality of HSPC within their vascular niche is strongly affected by interaction between local MSC and macrophage (Chow, et al. 2011) . BP stimulates oxidative stress mediators and inflammatory cytokines within 6h ( Figure 1C ), probably from BM macrophage (Chow, et al. 2011) . Il1b is increased 5-fold within 6h, before declining to initial levels after 12and 24h ( Figure 1C ). Tnf and Ccl3 are similarly responsive ( Figure 1C ). This cytokine response and the selective BP protection mechanism each depend on AhR and CYP1A1 (Larsen, et al. 2016; N'Jai A, et al. 2011; Rondelli, et al. 2016) . DMBA does not produce this cytokine response (Larsen, et al. 2016; N'Jai A, et al. 2011; Rondelli, et al. 2016) . CYP1A1 additionally converts BP to quinones, as major products (Keller and Jefcoate 1984) , which cause ROS activation of macrophage and cytokine release (Bolton and Dunlap 2017) .

DMBA has no equivalent product since the radical-cation pathway that generates BP quinones (Chakravarti, et al. 2008) converts DMBA to the 7-and 12-hydroxymethyl derivatives (Gehly, et al. 1979) . DMBA 8,9-o-quinone forms from secondary dehydrogenation of 8,9-dihydrodiol (Penning 2014) , but too slowly to deliver protective cytokines. The HSPC factor, Cxcl12, increases as the cytokines decline, whereas Csf1 and Pdgfa remain constant ( Figure 1C ).

Cells that are eluted from BM include large, extended fibroblastic cells that adhere to plastic and sequester a ten-fold larger number of hematopoietic cells, including the HSPC that deliver J o u r n a l P r e -p r o o f colony expansion (N'Jai A, et al. 2011) . Culture of this mixed population for 3 weeks expands the MSC population, while increasing the basal CYP1B1 content by 100-fold and largely removing induction by PAHs (Heidel, et al. 1998; Phinney, et al. 1999) . The BMS2 cell line, selected from this expansion, provides an informative model of MSC. The enrichment of MSC is readily tracked through the increased proportion of adherent cells that undergo adipogenesis (Hu, et al. 2018) . The BMS2 cells exhibited cell rounding and lipid droplet formation when treated for 8d with an adipogenic cocktail ( Figure 1D ). This response is replicated in adherent BM cells, but only after 7 days cell expansion that enriches MSC ( Figure 1D ). This is consistent with the enrichment of MSC over the course of the 15-day culture period (Phinney, et al. 2005; Phinney and Prockop 2007) .

The adipose markers are, however, mixed with osteoblast markers in the these cells, much as reported for C3H10T1/2 cells (Kelly, et al. 1998) . These MSC lines produce adhesion and adipogenic responses that are each blocked by a combination of AhR activation (TCDD) and

Mek-Erk stimulation (Egf or Fgf) (Hanlon, et al. 2005a; Liu and Jefcoate 2006) . The HSPC CFU activity is sustained in adherent BM cells by co-culture with BMS2 cells ( Figure 1E ). This support is further reproduced by media alone that is enriched over 24h with the cells ( Figure   1E ). DMBA inhibition is reproduced in vitro from CYP1B1 activation in the MSC cells (Rondelli, et al. 2016) .

The BMS2 cells express much higher high levels of basal CYP1B1 than C3H10T1/2 cells, as shown by either the 5.2 kb mRNA or the immunoblotted protein ( Figure 1F , left and right, respectively). This basal protein shows only modest induction by AhR activation (TCDD treatment). Basal expression is sustained in enriched AhR-ko primary BM (BM/6J AhR-/-) cells, but is completely removed in basal mouse embryo fibroblasts (Heidel, et al. 1999) . BMS2 and primary BM cells are devoid of CYP1A1 protein, even though inducible mRNA is detectable.

The CYP1B1 metabolic activity in the MSC lines mediates the in vitro DMBA suppression.

participation in BM microvascular niche MSC.

Single cell sequencing of adherent BM cells points towards multiple MSC types, including those marked by expression of the leptin receptor (Lepr) (Lepr+MSC) (Severe, et al. 2019; Tikhonova, et al. 2019) . These single cell expression profiles overlap appreciably with BMS2 cells (Tables 2C, S5 and S6). MSC have been estimated to represent about 0.3 percent of BM cells (Zhou, et al. 2014) . We confirm this assessment, here, with highly expressed markers, such as Cxcl12 and Csf1. MSC that match BMS2 expression of these markers are present at about 3 percent of adherent BM cells, which in turn represent about 10 percent of total eluted BM cells (Boregowda, et al. 2016; N'Jai A, et al. 2011) . Lepr+MSC, which represent a high proportion of these MSC (Severe, et al. 2019) , have been positioned to distinct BM niche vascular compartments with fluorescent markers and enriched for more in depth sequencing (Tikhonova, et al. 2019) .

presents three ways that we have used BMS2 cells to assess how CYP1B1 functions in MSC within the hematopoietic niche: 1) characterize the selectivity of BMS2 gene responses to DMBA and BP. Compare these responses to those derived from equivalent in vivo PAH treatments by using rapidly isolated adherent BM cells; 2) evaluate the anomalous BMS2 constitutive AhR regulation of CYP1B1, notably the high constitutive expression and low PAH induction; 3) examine the overlap of BMS2 mRNA profiles with those of the recently reported single cell clustering of Lepr+MSC populations (Severe, et al. 2019; Tikhonova, et al. 2019) .

This overlap tests whether there is a core set of functional markers that are conserved, in contrast to adaptive clusters and shifts in the poise between diverse mesenchymal differentiation fates.

CYP1B1 induction through AhR activation is a key feature of the PAH-mediated response in most cell types (Li, et al. 2017) . In BMS2 cells, the appreciable basal CYP1B1 gene expression is unaffected by DMBA over a 24h period (Figures 1F, left and 3A ). CYP1A1 has insignificant basal expression, but is induced to almost 40 percent of CYP1B1 levels after a 24h stimulation ( Figure 3A) , while failing to yield detectable protein ( Figure 1F , right) (Heidel, et al. 1999) .

Two other genes with canonical DRE elements, Aldh3a1 and Tiparp, show peak stimulations after 8h ( Figure 3A) . The TCDD-mediated stimulation of AhR-responsive genes in C3H10T1/2 cells also reached maximum induction in 8h (Hanlon, et al. 2005b) . Suspected stress response genes (Ptgs2, Cxcl10, Cdkn1a/p21 and Ccng/cyclin G1) failed to respond to DMBA during the first 8h but showed a secondary response to DMBA between 12-and 24h ( Figure 3B ).

Cdkn1a/p21 and Ccng1/Cyclin G1 are both well characterized cell cycle responses to p53 activation (Reinke and Lozano 1997) . These genes do not respond to TCDD or to BP ( Figure   3C ).

Hierarchical clustering of these gene responses, selected for preferential 24h responses Tables 1A-C, S2 and S3 provide a more quantitative perspective of the preferences of BP and J o u r n a l P r e -p r o o f DMBA for, respectively, direct AhR activation and metabolite driven responses. DMBA also shows a striking synergy between the two processes. Each of these tables includes the 8h response to TCDD, which defines the optimal direct AhR stimulation, without effects from metabolites. Table 1A shows direct 8h gene responses to DMBA and BP that parallel TCDD stimulations, with 12 genes exhibiting direct PAH responses, 5 of which have well established DREs (CYP1A1, Ahrr, Aldh3a1, Tiparp, and Nqo1) (Lee, et al. 2015) . BP stimulations paralleled TCDD stimulations, while DMBA was less active.

Notably, DMBA compensated with further substantial increases (>30%) between 8-and 24h (11/12 genes). Table 1B shows the prevalence of this biphasic PAH stimulation, which is also seen for canonical AhR responders, CYP1A1 and Ahrr (Table 1A) . This synergy of direct AhR activation, with delayed metabolite stress, is the prevailing mechanism for DMBA. Many AhRresponsive genes carry additional elements for factors that respond to chemical stress (NFB, p53, and Nrf2) (Kalthoff, et al. 2010; Mitchell and Elferink 2009; Tian, et al. 1999; Tijet, et al. 2006; Wakabayashi, et al. 2010) . The consistency of the finding suggests that AhR/ARNT activity is further activated at a single element. This overlap is most extensive for partnership with Nrf2 (Nault, et al. 2018) .

Comparison of these 12 gene responses to TCDD in C3H10T1/2 cells shows similar shared 8h stimulations, including CYP1A1, Aldh3a1, Adh7 and Nqo1 (Hanlon, et al. 2005a; Hanlon, et al. 2005b ). There are also AhR targets in C3H10T1/2 cells that are not repeated in BMS2 cells (Ch3l1, Glypican 1 and Sod3).

DMBA-mediated metabolic activation between 8-and 24h is much more effective than for BP. J o u r n a l P r e -p r o o f Table 1C shows 25 highly expressed genes that were stimulated by DMBA after 24h, but not after 8h. Each increase has been previously linked to p53 stimulation (Table S1 ). An additional 22 DMBA-selective genes are shown in Table S2 , several associated with p53 activity. None of these genes were stimulated by TCDD. Many of the genes in Table 1C respond fully to p53 within 8h, when DSB are produced directly by γ-radiation (Fei and El-Deiry 2003) . The slower DMBA response probably arises from delayed PAHDE generation (Keller, et al. 1987) . Only 14 genes showed a preferential stimulation by BP (FC>1.8) ( Table S3) . A further 7 showed equal responses to BP and DMBA. Suppression was also produced after 8h, again with preference for DMBA (Table S4) .

There was only a small preference for CYP1B1-mediated DMBA metabolism (24h), relative to BP metabolism, in BMS2 cells, assessed in the general cell activation of 15S p53 (Fei and El-Deiry 2003; Mirzayans, et al. 2013) (Figures 3E and S1A) , despite the large DMBA preference for gene effects mediated by p53 (Table 1C) . p-H2AX, another more direct marker of ATM kinase activation, showed a similar modest preference for DMBA compared to BP ( Figure 3E ). This may result because the distorted DMBA 3,4-dihydrodiol-1,2-epoxide structure is more active than the corresponding more planar BP 7,8-dihydrodiol-9,10-epoxide in the context of gene chromatin structure (Chakravarti, et al. 2008; Dreij, et al. 2005) . This incell immunodetection method was validated by the selective inhibition of this p53 activation by specific CYP1B1 inhibitors, TMS and -NF, in CYP1B1-V79 cells, but not CYP1A1-V79 cells (Figures S1B and C) .

Although the basal expression of CYP1B1 in primary MSC from BM or from MEFs depends on J o u r n a l P r e -p r o o f AhR (Alexander, et al. 1997; Heidel, et al. 1998) , the low PAH induction in BMS2 cells may derive from unusually high basal AhR activity. AhR induction of CYP1B1 is completely dependent on two highly conserved tandem SP1 sequences and a 265 base Ah-receptor enhancer region (AhER) (Wo, et al. 1997; Zheng and Jefcoate 2005; Zheng, et al. 2013) . This AhER has three AhR/ARNT complexes working in concert, two of which are substantially suppressed by an overlapping AhR inhibitory complex (AIC) (Zhang, et al. 1998; Zhang, et al. 2003) .

ChIP analyses showed similar basal AhR binding to the CYP1B1 and CYP1A1 promoters in mammary cells, despite the selective basal expression of CYP1B1 (Yang, et al. 2008) . Cellselective basal signaling evidently contributes to the consistent basal CYP1B1 expression.

Repression of basal AhR signaling by cell adhesion was shown in C3H10T1/2 cells and bcatenin released from adhesion complexes was shown to be a basal participant (Cho, et al. 2004) . This participation of WnT/b-catenin signaling in basal CYP1B1 expression has been further established in multiple cell types (Mohamed, et al. 2019; Ziegler, et al. 2016) . A further consideration is that cell selectivity for CYP1B1 and CYP1A1 in C3H10T1/2 and Hepa cells is primarily determined by selective chromatin interactions that are removed by DNA methylation (Beedanagari, et al. 2010a; Beedanagari, et al. 2010b) .

We further examined the basal and induced signaling to CYP1B1 in BMS2 cells with a luciferase reporter comprised of the dual SP1 elements linked to the AhER. Key elements in the AhER are three 12 base sequences (XRE 1, 4 and 5) that each bind the AhR/ARNT complex ( Figure 4A ) (Zhang, et al. 1998; Zhang, et al. 2003) .Two matched XRE's (1 and 4) are less active due interaction with the AIC (Zhang, et al. 2003 ).

An AhR antagonist, 3MNF (Ryan, et al. 2007) , removed most basal CYP1B1 protein expression J o u r n a l P r e -p r o o f ( Figure 4B ) and reduced the basal AhER/SP1 reporter activity by three-fold ( Figure 4C) . The basal inhibition progressed up to 0.1 uM, but surprisingly reversed at higher concentrations (0.1-10 M), suggesting alternative binding sites. Low TCDD induction of CYP1B1 in BMS2 cells is matched by the AhER/SP1 reporter. The incomplete suppression of basal expression by MNF is consistent with the appreciable expression in AhR-/-primary BM cells. The AhER/SP1 reporter showed low basal and the strong induction in C3H10T1/2 cells, thus paralleling the protein expression (Figures S2A and B) .

We tested the AhER-SP1 interactions in basal and induced cells by introducing mutations that blocked AhR binding to each of the XREs (Figure S2C) . These changes had similar effects on basal and induced activities in BMS2 and C3H10T1/2 cells (Figures 4D and S2D) . The XRE5 is essential for activity (MXRE5). Mutation of the distal XRE1 (MXRE1) and of XRE4 (MXRE4) are ineffective individually, but the double mutation fully removes expression (Zhang, et al. 2003) .

Mutations of the XRE4 (MXRE4), the Ebox (MEBOX) and their double mutation (MEBOX/MXRE4) indicated a partnership between these adjacent complexes (Figures 4D and   S2D ).

We also simplified the AhR enhancer by using a tandem quadruple XRE5 repeat (4XRE5), which then removes participation by the AIC (Figure 4A) . The basal and induced activities are appreciably higher (Figure 4D) , consistent with removal of the AIC. The high basal activity in CYP1B1 in BMS2 cells, therefore, derives from effects of cell-selective factors on the SP1/AhR partnership.

CYP1B1 transcription in C3H10T1/2 cells showed important contributions from cell adhesion, cell density and EGF signaling, each involving AhR participation (Cho, et al. 2004; Cho, et al. J o u r n a l P r e -p r o o f Journal Pre-proof 2005) . For AhER and 4XRE5 reporters in BMS2 cells, density increase from 50 to 80 percent shows large increases in basal activity, but not in TCDD induction ( Figure 5A) . However, at 90 percent, additional cell-cell contacts cause both activities to decline. This increase in basal density-dependent signaling is consistent with increased effects of secreted cell factors. This signaling overlaps the effect of TCDD on the AhR, which is, therefore, not further stimulated.

Enhanced cell-cell contacts block both autocrine and TCDD signaling. This suppression by cellcell adhesion appears as the reverse of the AhR-dependent stimulation when adhesion is disrupted in C3H10T1/2 cells (Cho, et al. 2004) .

For CYP1B1 and CYP1A1, mRNA expression induction, but not basal expression, was elevated by an increase in cell density ( Figure 5B) . The natural genes are more responsive to ligand-free AhR than the reporters, but less responsive to TCDD-activated AhR, particularly at the low density. At 80 percent of confluence, the autocrine boost enhances the TCDD-activated AhR more than the basal expression. At this density, the reporters and genes then respond similarly.

CYP1A1 also responds similarly, suggesting that AhR is directly affected. The opposing effects of cell density and cell contacts suggests that the balance of effects may differ between chromatin-free reporters and the histone sequestered gene. C3H10T1/2 cells showed similar trends of cell density for reporters and natural CYP1B1 ( Figure S2E ). Low activities in confluent C3H10T1/2 cells are activated by removal of cell-cell contact, in parallel with nuclear translocation of the AhR (Cho, et al. 2004) .

We also showed that co-culture with BMS2 cells elevated basal reporter activity in C3H10T1/2 cells ( Figure 5C ). BMS2 basal activity declined. Thus, BMS2 cells release net activators, while the C3H10T1/2 cells release net suppressor factors. Again, the maximum TCDD-induced activities are unaffected. Thus, autocrine/paracrine factors activate the AhR partnership with SP1 to maximum levels produced by TCDD complex formation.

Cxcl12, a MSC participant in HSPC support that is also regulated by SP1 and adhesion (Schajnovitz, et al. 2011) , showed an even larger cell density stimulation effect in BMS2 cells ( Figure 5D) . Csf1, another HSPC stimulant, was unaffected ( Figure 5D ). This density increase in Cxcl12 was also shown in C3H10T1/2 cells (Figure S2E ).

A search of the BMS2 transcriptome for secreted activator proteins and paired receptors identified eight pairs with potential for autocrine regulation ( Table 2A) . The asymmetry of gene expression between secreted factors and their cognate receptor is repeated across six distinct Lepr+MSC clusters (Tikhonova, et al. 2019) . Thus, the paracrine pairings Pdgfa/Pdgfrb, Fgf7/Fgfr2 are retained as potential autocrine stimulants of Mek/Erk (Andrae, et al. 2008; Chen, et al. 2013; Noriega-Guerra and Freitas 2018) . In C3H10T1/2 cells, EGF activates Mek-Erk and stimulates AhR induction of CYP1B1 by TCDD (Hanlon, et al. 2005b ). TCDD also functions in concert with Mek-Erk to enhance focal adhesion signaling ,thereby to suppress MSC adipogenic differentiation (Hanlon, et al. 2005b ). SP1 is phosphorylated through this pathway suggesting a likely activation mechanism (Karkhanis and Park 2015) .

Macrophage cytokines show striking asymmetry in MSC expression. They are largely absent in the BMS2 cells, while their cognate receptors are highly expressed (Tnfrsf1a, Ifngr1, Il1r1) ( Table 2A) . Ccl2 and Ccl7 are retained as the only C-C chemokines but, again, with absence of their receptors (Ccr2). Many of these paracrine receptors correspond to cytokines that are highly stimulated by BP, in vivo, within 6h ( Figure 1C) . This asymmetry of expression supports a model in which BP-stimulation of cytokines in niche macrophage produce changes in MSC signaling that may contribute to stress protection. Thus, macrophage stimulation can potentially contribute to a selective redistribution of the clusters.

The third asymmetric display comprises paracrine donors that target other cells, as the BMS2 cells lack endogenous expression of the paired receptor (Table 2A) . For example, Kitl, which controls HSPC, is expressed without the corresponding c-Kit receptor. Likewise, Ccl2 functions as an angiogenic and immune stimulant (Lim, et al. 2016) . These pairings are highly selective among the clusters.

In Table 2B , we compare expression of abundant functional genes from BMS2 cells to their expression in adherent BM cells. mRNA from MSC represents less than 5 percent of the total in this fraction, which largely derives from hematopoietic cells attached to the MSC (Phinney and Prockop 2007) . These highly expressed BMS2 genes (Cy3>3500) and adherent BM mRNA are ranked by proportion of the BMS2 content (Supplement Excel file). Approximately 100 genes appear in the 1-6 percent range, including CSF1 (4 percent), Gas6 (2 percent), Ccl2 (6 percent), Cxcl12 (3 percent), Kitl and Ebf3 (1-2 percent) ( Table 2B) . Each has been linked to MSC activity, thus suggesting that they are markers for BMS-like cells within the adherent BM-cell fraction (at 3 percent of the content).

Several matrix proteins, including Col1a1, Col1a2, Col2a1 and Fn1 are also expressed in this range, but CYP1B1 and a substantial proportion of matrix proteins and regulators (Fbln2, Ctgf, Wisp2) have relatively far higher expression in BMS2 cells ( Table 2B ).

The continuous enrichment of primary MSC, which precedes the generation of BMS2, is enabled by matrix processes that may facilitate this enrichment. CYP1B1 protects vascular cells from oxygen-induced stress, which is generated by culture under ambient oxygen (20 percent) conditions (Palenski, et al. 2013b; Rondelli, et al. 2016) . Select genes, including three exosome J o u r n a l P r e -p r o o f markers, Lpl and Thbs1 (thrombospondin 1), show high expression in both adherent BM and BMS2 cells (Table 2B) , likely reflecting alternative sites of expression in adherent BM cells.

The genes with BMS2/adherent BM overlap appropriate to MSC expression were further compared to the single cell profile of Lepr+MSC and other vascular niche cells (Severe, et al. 2019) (Table 2C ). This cluster of cells express CYP1B1 as one of the 30 most abundant mRNA (N>300). Lepr is absent from BMS2 cells ( Table 2C) , but is also low in other MSC clusters (Severe, et al. 2019) . Twelve highly expressed BMS2 genes and CYP1B1 show specificity for the Lepr+MSC ( Table 2C) . Each of the 12 markers has a functional basis for a presence as a core MSC gene (Figures 6A and S3) . The expression of these BMS2 mRNA markers correlated with their expression in BM adherent cells (r 2 = 0.86) ( Figure 6B) .

The expression levels for 28 genes from BMS2 cells have matches among the most highly expressed genes identified by single cell sequencing of Lepr+MSC in BM (Table S5 ) (Severe, et al. 2019) . BMS2 cells, like Lepr+MSC, also express markers for differentiated mesenchymal cell types in the BM niche (Table S6) , including osteoblast-like cells (OLC), a chondrocyte (Chond) and a fibroblast-like cluster (Fib) as well as representative endothelia (EC) and pericytes (Per). Several of the MSC expressed genes are highly expressed in OLC, but less than in Lepr+MSC. High expression of Vcam1 and Pdgfrb is shared by MSC and, respectively, EC and Per (Table S5) . We also show extensive overlap of cytokine receptors between Lepr+MSC and the other cell types from the BM vascular niche. The distribution of these receptors among different cell types matches the broad diversity of cytokines that are generate by BP (Figure 1C ).

The improved availability was provided by Lepr+ enrichment, which delivered six distinct populations (P1-5 and C) of Lepr+MSC (Table S7) (Tikhonova, et al. 2019) . Some of the Lepr+MSC clusters show expression biases to either adipoblast or osteoblast differentiation. We used the supplementary single sequencing data of these Lepr-GFP select clusters to show that CYP1B1 expression was highly correlated not only with Lepr, but also with Cxcl12, Kitl, Csf1 and Gas6 (Figure 6B, Table S7 ). Ebf3, Ptx3 and Il1rn additionally have high expression in the C cluster (proliferative cluster) and, thus, have weaker correlation with the other core genes.

IL7, Fzd1, Svep1 and Nnmt have relatively low expression in one cluster (P1), which is specifically marked by Ccl2 and Ccl7. IL7, the major lymphoid stimulant, demonstrates low expression in the adherent BM fraction and BMS2 cells and only appears as a core gene after Lepr+ enrichment. Spp1 marks P4 and osteoblast clusters. Lpl, an adipocyte marker, is selectively expressed in P2 and P5.

Even though DMBA mediates extensive suppression of HSPC expansion (Figure 1A) , which we previously identified as dominated by cytokines ( Figure 1C ).

This absence of stress changes in the cultured BM cells that are seen in BMS2 cells is consistent with stress responses that are not broadly distributed among the total BM population.

They are local to the niche presence of MSC and CYP1B1, which our data indicates to be only about 3 percent of adherent cells. Thus, only highly expressed stress response markers are to J o u r n a l P r e -p r o o f be expected. Ccng1, Sulf2, Phlda3 (each p53-induced, Tables 1C and S1) are retained the DMBA stimulation seen in BM cells (Figure 6C) . Other genes that showed strong p53 stress responses to DMBA in BMS2 cells (Gdf15, Gadd45a and Txnip) ( Table 1C ) did not respond but were predominantly expressed in other adherent cells types (data not shown).

Highly expressed markers shared by BMS2 cells and Lepr+MSC (Cxcl12, Csf1, Gas6) ( Table   2C ) were scarcely affected by DMBA in the BM cells. Ccl2, Spp1 and Lpl, which each mark more polarized MSC clusters (respectively, P1, P4 and P5; Table S7 ), show significant increases, as if stress is redistributing the Lepr+MSC (Figure 6C ).

PAHs produce diverse effects on the immune system, particularly on T-lymphocytes and dendritic cells (O'Driscoll, et al. 2018; O'Driscoll and Mezrich 2018) . PAHs activate the AhR, which regulates the balance between effector and regulatory T cells (O'Driscoll and Mezrich 2018) and induces CYP1A1 and CYP1B1. Opposing effects of PAHs on HSPC, typified by DMBA and BP and mediated by, respectively, CYP1B1 and CYP1A1, occur in BM within 6 hours (Figure 7: Paths A and B) . Both focus on the support of HSPC by Lepr+MSC within the BM vascular niche. The BMS2 cells line effectively models critical roles by CYP1B1 and other regulatory proteins in the Lepr+ MSC.

Path A requires effective activation of the PAH, typified by CYP1B1-mediated metabolism of DMBA to PAHDE in MSC, which extensively suppresses HSPC and, within 48h, mature lymphocytes in BM, thymus and spleen (Figure 1) (Larsen, et al. 2016) . In the BM, HSPC, including lymphoid progenitors, are controlled by factors released from Lepr+MSC within the vascular niche (Agarwala and Tamplin 2018; Seike, et al. 2018; Severe, et al. 2019; Tikhonova, et al. 2019) . Factors from BMS2 cells sustain HSPC freshly isolated from the BM (Figure 1 ).

Metabolism of DMBA by CYP1B1 in these MSC removes lymphoid progenitors (Rondelli, et al. 2016) . DMBA metabolism in BMS2 cells does not remove support factors, but instead generates PAHDE, which produce DNA adducts, DSB and associated p53 activation in MSC and adjacent HSPC (Heidel, et al. 2000) . Primary BM MSC and BMS2 cells express high basal levels of CYP1B1, with unusually weak AhR activation (Figures 1 and 3) , even though other AhR marker genes are extensively induced by TCDD, BP and DMBA (Figures 3 and 6, Tables 1A-C and   2C ).

In vivo, a second type of PAH activity, typified by BP, completely prevents this suppression of HSPC (Figure 7: Path B) . This protection arises from systemic metabolism by CYP1A1 from outside the vascular niche, probably the liver (Larsen, et al. 2016; N'Jai A, et al. 2011; N'Jai A, et al. 2010) . The BP protection is completely dependent on AhR (N'Jai A, et al. 2011) . BP protection matches an extensive AhR-and CYP1A1-dependent stimulation of cytokines (IL1b, TNF and IFNg) from macrophage in the BM within 6h (Figure 1) . CYP1A1 is scarcely present in BM or BMS2 cells (Figure 1 ), but AhR induction by PAHs yields high levels in the liver (Galvan, et al. 2005) . Thus, in vivo BP metabolism and circulating metabolites, including quinones, peak within 6h, just as BM cytokines appear (Figure 1) (Larsen, et al. 2016; N'Jai A, et al. 2011) .

Equivalent quinones are not matched for DMBA (Gehly, et al. 1979) . Radiation-induced ROS in macrophage initiates NFkB activation that increases similarly high levels of IL1b (Bigildeev, et al. 2013) . PCB quinone stimulation of RAW 264.7 macrophage generates p65 NFKB phosphorylation that activates the same set of cytokines (Yang, et al. 2019) . Il1b stimulates proliferation of MSC via the Il1 receptors and activates over 400 genes (Amann, et al. 2019) . BP effects on BM produce other increases (Il6, Ccl7, Cxcl1) (Larsen, et al. 2016; N'Jai A, et al. 2011 ) that match reported direct stimulation of MSC by IL1b (Hengartner, et al. 2015) . BMS2 cells effectively model Lepr+MSC, which also express substantial CYP1B1 (Figure 6 (Table 2A) . This cluster of 12 key genes link to BMS2 cells in three ways: the expression overlap between BMS2 and adherent BM expression (Figure 6) ; their shared prevalence in BMS2 cells and in a Lepr+MSC cluster derived from a total BM single cell transcription (Tables 2C, S5 and S6) (Severe, et al. 2019) ; and their close correlation across Lepr+-enriched MSC clusters in BM (Table S7 ) (Tikhonova, et al. 2019) .

In both Lepr+ selections, CYP1B1 emerges as an equal member with the other 12 genes (Tables 2C, S5, S6, S7) . The lymphoid progenitor stimulant, IL7, has low expression, but emerges as a cluster member after the Lepr+ enrichment (Tikhonova, et al. 2019) . These relationships point to a cluster of BMS2-like cells that are present at 3 percent of adherent BM cells and 0.3 percent of total BM content. The low content of CYP1B1 in adherent BM cells compared to BMS2 cells probably derived from selective enrichment driven by the protection from oxygen stress that is provided by CYP1B1 (Palenski, et al. 2013a) . Equivalent adaptive enrichments in BMS2 expression likely apply to several ECM-associated genes.

In BMS2 cells, CYP1B1 metabolism of DMBA that produces PAHDE adducts (Heidel, et al. 2000) also stimulates numerous genes, but only after 8h (Tables 1A-C and S2). About half have previous reported evidence for p53 activation (Tables 1C andS1) , which is a necessary part of the HSPC suppression process (Page, et al. 2003) . BP is much less effective but is more active in direct 8h AhR activations (Table 1A) . For DMBA, the smaller direct AhR activation, is almost invariably boosted by a second phase of metabolite activation, even for CYP1A1 (Table 1A, Figure 3 ). This second phase is likely to derive from supplementary Nrf2 activity on the J o u r n a l P r e -p r o o f same AhR/ARNT elements (Nault, et al. 2018) .

This selectivity by DMBA is enhanced by the chromatin environment of the gene since DMBA and BP were similarly effective in the total cell activations of H2AX and p53 (Figure 3) . Gene selectivity for individual PAHDE is structure dependent (Chakravarti, et al. 2008; Dreij, et al. 2005) . The 8h delay almost certainly arises from the time to generate the PAHDE (Keller, et al. 1987) , since the same genes are maximally activated by gamma-radiation in 8h (Fei and El-Deiry 2003; Mirzayans, et al. 2013) . Table 1C) but are far too slow to mediate the 6h progenitor suppression times that are produced in the Lepr+MSC the BM niche (Figure 1) . Evidently, more direct effects of DMBA metabolites produce faster, non-transcriptional steps (Pietrocola, et al. 2013) . For example, apoptosis initiated through Bcl2 in the mitochondrial outer membrane (Vaseva and Moll 2009 ) is a favored p53 intervention in stem cells (TeSlaa, et al. 2016) . The observed 48h recovery from DMBA toxicity (Figure 1 ) may, however, derive from this p53-mediated transcription. Most of the BMS2 genes that respond to DMBA (Tables 1A-C, S1 and S2) are insufficiently expressed to be detectable in MSC at 3 percent of the adherent BM cells. Only three increases (Ccng1, Sulf2 and Phlda3) parallel activations seen in BMS2 cells (Figure 6, Tables 1B and C) . Ccng1 is a potential marker for p53 activation in BM MSC (Reinke and Lozano 1997) . Ccng1 is highly selective to DMBA in both BMS2 cells (Figure 3, Table 1C ) and adherent BM (N'Jai A, et al. The novel, high constitutive CYP1B1 expression in BMS2 cells (Figure 1) is predominantly controlled by a strong coupling between the three AhR/ARNT complexes on the DRE of the AhER and the dual SP1 repeats close to the transcription start site (Figures 4 and 8) . In the basal state, an autocrine boost by growth factor signaling to SP1 is sufficient to avoid external AhR activation. This cooperation may be enhanced by release of catenin from cell junction sites (Cho, et al. 2005; Ziegler, et al. 2016) . Cell-cell adhesion may suppress CYP1B1 expression by withdrawing catenin from the nucleus to the junction sites. SP1 is an important regulator of MSC, including for CXCL12, which parallels CYP1B1 in the cell density stimulation ( Figure 5) . SP1 is commonly activated by Erk phosphorylation, including through activation by Pdgfa (Gong, et al. 2017; Schajnovitz, et al. 2011) . This Pdgfa/Pdgfrb pairing is highly expressed, but Hgf/Met and Fgf7/Fgfr2 pairing also activate Erk (Figure 8) . Cxcl12, Tgfb3 and Wnt2 provide additional pairings. Wnt2/Fzd1 activates -catenin, possibly participating in the adhesion associated AhR changes.

The strong correlations of CYP1B1 with the other 12 MSC markers (Figure 6 ) suggest a physiological role in the MSC regulation of the vascular niche. Other vascular roles include estradiol hydroxylation (Dempsie, et al. 2013; Pingili, et al. 2017) , epoxidation of polyunsaturated fatty acids (Lefevre, et al. 2015; Li, et al. 2015) and suppression of vascular oxidative stress (Palenski, et al. 2013a ).

In summary, two distinct but opposing effects of PAHs have been identified that impact MSC control of HSPC within the BM vascular niche (Graphic Abstract, Figure 7) . Path A suppression is mediated within 6h by high levels of CYP1B1 and Ephx1 in Lepr+MSC that generate PAHDE. DMBA and environmental PAHs that each generate sterically hindered PAHDE are most active (Chakravarti, et al. 2008) . The high basal CYP1B1 in BM MSC result J o u r n a l P r e -p r o o f from partnership between AhR and SP1, boosted by autocrine signaling (Figure 8) . Path B protection derives from PAH quinones, delivered to BM by hepatic CYP1A1 metabolism, which in turn depends on PAH induction via AhR. These products depend on radical cation generation, which is typical for other multi-ring PAHs (Benz(a)anthracene, Chrysene). AhR activation is, however, higher for BP than most other PAH, including DMBA. The mouse AhR genotype is a major factor (N' Jai A, et al. 2011) , with strains divided between PAH-responsive AhRb and resistant AhRd alleles, due to binding site sequence variation (Seok, et al. 2018 ) that, however, does not affect constitutive activity. In AhRd strains, BP provides no protection and indeed delivers enough PAHDE to match DMBA as a suppressor (N'Jai A, et al. 2011) .

The mode of protection by PAH quinones probably derives from Il1b, which is stimulated in BM macrophage. Stimulation of MSC through their IL1R receptors (Bigildeev, et al. 2013) could, for example, attenuate the ATM/p53 response to PAHDE. We have identified BM increases that match with IL1b/Il1R activation of human MSC (Hengartner, et al. 2015) . Thus, BM increases in IL6 and Ccl7 track with IL1b increases (Figure 1, Table 2A) . We can now test effects of IL1b on the DMBA p53-mediated responses in BMS2 cells or the HSPC/BMS2 co-culture model.

To assess environmental exposures on BM vascular niches, the effects of CYP1B1/Ephx1 generation of PAHDE in BMS2 can now be compared to PAH quinone stimulation of IL1b and other cytokines in BM macrophages or Raw 264.7 cells (Bolton and Dunlap 2017; Yang, et al. 2019) . V79 cells that express human CYP1B1 and CYP1A1 provide a means to probe species shifts in metabolic selectivity (Luch, et al. 1998; Luch, et al. 1999; Schmalix, et al. 1993) .

Environmental exposure impacts AhR induction of CYP1A1 and, thereby, metabolic increases in the flux of PAH quinones to the BM macrophage that then stimulate cytokine production.

Combustion pollution mixtures also include diverse PAH quinones and AhR activators (Bostrom, et al. 2002; Layshock, et al. 2010) .

J o u r n a l P r e -p r o o f BMS2 cells and Lepr+MSC share CYP1B1 and a very limited core of Lepr+MSC functional markers as well as a set of cytokine receptors that complement the macrophage cytokine production (Figure 6, Tables 2A and C) . CYP1B1 expression in Lepr+MSC is highly correlated with four secreted HSPC regulatory factors (Cxcl12, Csf1, Kitl, Svep1 and Il7), two secreted immune modulators (Gas6, Ptx3) and FZD1, which directs Wnt2 activity (Figure 6) . These genes are notably very resistant to Path A and Path B signaling. BMS2 cells express many additional genes that appear to be gained from in vitro selection. The effectiveness of OP9/BMS2 co-culture with BM HSPC (Rondelli, et al. 2016) provides confidence that they indeed model PAH effects on Lepr+MSC.

Highlights  BMS2 and Lepr+MSC cells co-express CYP1B1 and 12 functional niche activity markers.

 CYP1B1 mRNA in BMS2 cells depends on activation of SP1 coupled to an AhR enhancer unit.

 DMBA metabolism by CYP1B1 activates p53 gene targets in BMS2 cells far more than BP.

 HSPC suppression by CYP1B1 generation of PAHDE requires rapid, non-genomic targets. Treatments were completed in triplicate. Only genes with expression differences, p-values<0.05 and Cy5-values>100 were analyzed.

E. In cell western analysis of BMS2 cells show significant p53 phosphorylation in response to 24h PAH t r e a t m e n t .

Top: a representative fluorescence image of PAH-mediated p53 and H2AX phosphorylation obtained in the high throughput 96-wellplate assay.

BP (left) and DMBA (right) significantly activate p53 phosphorylation in BMS2 cells.

Significance (*) was defined as PAH-mediated fold change p<0.05 relative to DMSO vehicle control. BMS2 cells show high AhR-mediated basal CYP1B1 expression and low induction by TCDD and PAHs. Basal CYP1A1 is undetectable, but highly inducible (40 percent of CYP1B1). Coculture of BMS2 with C3H10T1/2 cells cause crossover stimulation of basal CYP1B1 in C3H10T1/2 cells, indicative of BMS2 release of secreted paracrine factors (SF) that stimulate C3H10T1/2 cells. Cell density effects suggest that SF contribute to the high basal CYP1B1 in MCL contributed to experimental design, completion of bone marrow isolation and microarray, adipogenic and CFU experimental analyses and writing of the manuscript.

AA completed bone marrow isolation and cell density experimental analyses.

TT completed bone marrow microarray analyses.

CMR contributed to writing and completed experimental analyses on control of HSPC activity by MSCs.

RJS completed the CYP1B1 promoter construct experiments.

CRJ completed the conceptual design and writing of the manuscript. 

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Ionizing radiation-induced responses in human cells with differing TP53 status

Timing is everything: consequences of transient and sustained AhR activity

Inflammatory breast cancer: Activation of the aryl hydrocarbon receptor and its target CYP1B1 correlates closely with Wnt5a/b-beta-catenin signalling, the stem cell phenotype and disease progression

Polycyclic aromatic hydrocarbons: from metabolism to lung cancer

Development of hematopoietic stem cell activity in the mouse embryo

Acute disruption of bone marrow hematopoiesis by benzo(a)pyrene is selectively reversed by aryl hydrocarbon receptor-mediated processes

Bone marrow lymphoid and myeloid progenitor cells are suppressed in 7,12-dimethylbenz(a)anthracene (DMBA) treated mice

Comparison of Hepatic NRF2 and Aryl Hydrocarbon Receptor Binding in 2,3,7,8-Tetrachlorodibenzo-p-dioxin-Treated Mice Demonstrates NRF2-Independent PKM2 Induction

Regulation of preB cell apoptosis by aryl hydrocarbon receptor/transcription factor-expressing stromal/adherent cells

Extracellular Matrix Influencing HGF/c-MET Signaling Pathway: Impact on Cancer Progression

Polycyclic aromatic hydrocarbons (PAHs) present in ambient urban dust drive proinflammatory T cell and dendritic cell responses via the aryl hydrocarbon receptor (AHR) in vitro

The Aryl Hydrocarbon Receptor as an Immune-Modulator of Atmospheric Particulate Matter-Mediated Autoimmunity

Cyp1B1 expression promotes angiogenesis by suppressing NF-kappaB activity

Lack of Cyp1b1 promotes the proliferative and migratory phenotype of perivascular supporting cells

Human aldo-keto reductases and the metabolic activation of polycyclic aromatic hydrocarbons

Murine mesenchymal and embryonic stem cells express a similar Hox gene profile

Plastic adherent stromal cells from the bone marrow of commonly used strains of inbred mice: variations in yield, growth, and differentiation

Regulation of autophagy by stress-responsive transcription factors

2017 2-Methoxyestradiol Reduces Angiotensin II-Induced Hypertension and Renal Dysfunction in Ovariectomized Female and Intact Male Mice

6beta-Hydroxytestosterone, a Cytochrome P450 1B1-Testosterone-Metabolite, Mediates Angiotensin II-Induced Renal Dysfunction in Male Mice

Characterization of a novel cytochrome P450 from the transformable cell line, C3H/10T1/2

Differential activation of p53 targets in cells treated with ultraviolet radiation that undergo both apoptosis and growth arrest

PAHs Target Hematopoietic Linages in Bone Marrow through Cyp1b1 Primarily in Mesenchymal Stromal Cells but Not AhR: A Reconstituted In Vitro Model

Environmental toxicants may modulate osteoblast differentiation by a mechanism involving the aryl hydrocarbon receptor

Mouse cytochrome P-450EF, representative of a new 1B subfamily of cytochrome P-450s. Cloning, sequence determination, and tissue expression

Recombinant mouse CYP1B1 expressed in Escherichia coli exhibits selective binding by polycyclic hydrocarbons and metabolism which parallels C3H10T1/2 cell microsomes, but differs from human recombinant CYP1B1

Mouse endometrium stromal cells express a polycyclic aromatic hydrocarboninducible cytochrome P450 that closely resembles the novel P450 in mouse embryo fibroblasts (P450EF)

CXCL12 secretion by bone marrow stromal cells is dependent on cell contact and mediated by connexin-43 and connexin-45 gap junctions

Stable expression of human cytochrome P450 1A1 cDNA in V79 Chinese hamster cells and metabolic activation of benzo[a]pyrene

Stem cell niche-specific Ebf3 maintains the bone marrow cavity

Trace derivatives of kynurenine potently activate the aryl hydrocarbon receptor (AHR)

Stress-Induced Changes in Bone Marrow Stromal Cell Populations Revealed through Single-Cell Protein Expression Mapping

Cytochrome P450 1b1 in polycyclic aromatic hydrocarbon (PAH)-induced skin carcinogenesis: Tumorigenicity of individual PAHs and coal-tar extract, DNA adduction and expression of select genes in the Cyp1b1 knockout mouse

12-dimethylbenz[a]anthracene-induced, stromal celldependent bone marrow B cell apoptosis: stromal cell-B cell communication and apoptosis signaling

Mitochondria in human pluripotent stem cell apoptosis

Ah receptor and NF-kappaB interactions, a potential mechanism for dioxin toxicity

Aryl hydrocarbon receptor regulates distinct dioxin-dependent and dioxinindependent gene batteries

The bone marrow microenvironment at single-cell resolution

Oral exposure to benzo[a]pyrene in the mouse: detoxication by inducible cytochrome P450 is more important than metabolic activation

Oral benzo[a]pyrene in Cyp1 knockout mouse lines: CYP1A1 important in detoxication, CYP1B1 metabolism required for immune damage independent of total-body burden and clearance rate

The mitochondrial p53 pathway

Aryl hydrocarbon receptor is required for optimal B-cell proliferation

EDGE(3): a web-based solution for management and analysis of Agilent two color microarray experiments

When NRF2 talks, who's listening?

Functional analysis of the promoter for the human CYP1B1 gene

Polychlorinated Biphenyl Quinone Promotes Macrophage-Derived Foam Cell Formation

Constitutive regulation of CYP1B1 by the aryl hydrocarbon receptor (AhR) in premalignant and malignant mammary tissue

Characterization of the mouse Cyp1B1 gene. Identification of an enhancer region that directs aryl hydrocarbon receptor-mediated constitutive and induced expression

Ah receptor regulation of mouse Cyp1B1 is additionally modulated by a second novel complex that forms at two AhR response elements

Steroidogenic factor-1 interacts with cAMP response element-binding protein to mediate cAMP stimulation of CYP1B1 via a far upstream enhancer

Stimulation of mouse Cyp1b1 during adipogenesis: characterization of promoter activation by the transcription factor Pax6

Leptin-receptor-expressing mesenchymal stromal cells represent the main source of bone formed by adult bone marrow

beta-Catenin Is Required for Endothelial Cyp1b1 Regulation Influencing Metabolic Barrier Function

We thank Dr. Owen Tamplin for his insightful discussions relating to the BM Lepr+MSC, which facilitated the identification of the 12 core Lepr+MSC genes in the BMS2 cell model. This publication was made possible by grants from the National Institute of Diabetes and Digestive and Kidney Disease (NIDDK) grant numbers DK072749 and DK090249, and the

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