*3.1. Genistein Does Not Exhibit Cytotoxic E*ff*ects on Primary Fibroblasts*

Primary B virus infection occurs at the epithelial layer of the skin and/or mucosa. Thus, we chose to test genistein's efficacy in primary human and macaque fibroblasts (HFF and RMF respectively). Genistein, in certain cell types, has been reported to inhibit cellular proliferation via cell cycle arrest, so we first verified that genistein would not have any cytotoxic effects on primary fibroblasts at doses used in this study. We tested the effects of genistein on cell viability, proliferation, metabolic activity, and DNA synthesis. Cell viability was measured at 48 h post-genistein treatment via trypan blue dye exclusion assay. Genistein had no notable effect on cell viability at 48 h post-treatment (Figure 1A, \B). Next we asked if genistein affected cell proliferation. Initially, we assayed cellular division in an asynchronistic population of cells and saw no effect (Figure 1C,D). We then tested if genistein's effect on cell doubling was masked in the asynchronistic population. Cells were synced in G0/G1 phase via serum-starvation, then released from cell cycle arrest by the reintroduction of serum with or without the addition of genistein. At all doses, the tested cells showed similar doubling times to the controls (Figure 1C,D). An alternative method of measuring cellular proliferation was performed using an MTS assay, which measures cellular metabolic activity via the cell's ability to breakdown tetrazolium dye or MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to insoluble formazan. Cells were serum-starved overnight then released from cell cycle arrest with the reintroduction of serum in the presence or absence of genistein. Cells were kept for either 48 or 72 h and then assayed for their ability to reduce MTT. Genistein did not reduce cellular metabolic activity in HFFs; in fact, an increase in metabolic activity was noted, particularly at median doses, which was most apparent after 72 h (Figure 1E). A slight, though insignificant increase in metabolic activity was also repeatedly noted in both cell lines following treatment with DMSO (vehicle control). In RMFs, genistein had a modest inhibitory effect at high doses with a 24% and 36% reduction in metaboloic activity at 50 μM and 100 μM, respectively, compared with the DMSO vehicle control (Figure 1F). When compared to untreated cells, the effect was less dramatic, with a 10% reduction in metabolic activity at 50 μM and 26% reduction at 100 μM. Finally, we assayed for the ability of cells to undergo DNA synthesis in the presence of genistein. Cells were brought into cell cycle arrest via serum-starvation, after which serum was added back in the presence or absence of genistein along with a thymidine analog, bromodeoxyuridine (BrdU), that is incorporated into newly synthesized DNA. There was no notable difference in the pattern of BrdU uptake in cells at any genistein concentration tested versus the controls [37]. Collectively, these data support that genistein is non-toxic to primary fibroblasts at the concentrations used in this study.

**Figure 1.** Cytotoxicity of genistein in human foreskin fibroblasts (HFFs) and Rhesus macaque foreskin fibroblasts (RMFs). Cells were left untreated (negative control) or treated with either 0.1% dimethyl sulfoxide (DMSO) (experimental control) or increasing concentrations of genistein. After 48 h, HFFs (**A**) and RMFs (**B**) were collected and assayed for live vs. dead cells via trypan blue dye exclusion. HFFs (**C**) and RMFs (**D**) were kept as an asychronous population or growth was synchronized via overnight serum-starvation prior to genistein treatment. Cells were collected and the total cell numbers counted after 48 h. HFFs (**E**) and RMFs (**F**) were serum-starved overnight, then genistein-treatments were added and cells were incubated for 48 h (**E**) or 72 h (**E**,**F**), and then assayed for metabolic activity via MTS assay. For A–F, *n* = 9, with the statistical analysis performed via one-way ANOVA with Dunnet's correction for multiple comparisons.
