Toxicity of ubiquitous tire rubber antiozonant N-(1,3-dimethylbutyl)- N'-phenyl- p-phenylenediamine (6PPD) and its transformation product 6PPD-quinone (6PPD-Q) in primary human hepatocytes and liver spheroids

Abstract

The tire rubber antioxidant N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine (6PPD) and its oxidation product 6PPD-quinone (6PPD-Q) were recently found in human bodies. Though 6PPD/6PPD-Q showed species-dependent toxicity in animals, human relevant data are scarce. Here, primary human hepatocytes (PHHs), the gold standard in vitro model for hepatotoxicity, were used for acute and subacute toxicity assessments, with test concentrations normalized to average human serum concentrations (C_ave). Acute exposure in sandwich cultured PHHs decreased glutathione starting at 100-fold C_ave of 6PPD (10 ng/mL) or 500-fold C_ave of 6PPD-Q (100 ng/mL), and inhibited albumin starting at 10,000-fold C_ave of 6PPD, or 2500-fold C_ave of 6PPD-Q. Urea was suppressed by 6PPD-Q, but not 6PPD, starting at 2500-fold C_ave. Lactate dehydrogenase (LDH) leakage, a measurement of cell death, was unaffected. Subacute exposure of primary human liver spheroids to 6PPD-Q showed no cell death, while 6PPD increased caspase 3/7 activity and LDH leakage and decreased adenosine triphosphate at 50,000-fold C_ave. Of 10 cytokines involved in hepatotoxicity, interleukin-8 was increased by 6PPD and 6PPD-Q starting from 200- and 50-fold C_ave, respectively. At 50 to 300-fold C_ave, the in vivo-relevant concentrations in humans, GSH, caspase 3/7 activity, and interleukin-8 were the only endpoints that were significantly affected by 6PPD and/or 6PPD-Q, and no cell death was observed. These data indicate that 6PPD/6PPD-Q may cause liver dysfunctions and trigger immunotoxicity in heavily exposed individuals but are unlikely to induce significant cell death at regular environmental exposure levels.

Keywords: 6PPD; 6PPD-Q; Liver injury; Primary human hepatocytes; Spheroids.

Graphical abstract

Fig. 1

LDH leakage from PHHs in response to 6PPD or 6PPD-Q. Sandwich-cultured PHHs were treated with 6PPD and 6PPD-Q for 3 days, and the relative levels of LDH activity in culture medium were measured using a luminescence kit (Promega). ∗p < 0.05 as compared to control. Data are means ± SDs (n = 4–10).

Fig. 2

Functional changes of PHHs in response to 6PPD or 6PPD-Q. Sandwich-cultured PHHs were treated with 6PPD and 6PPD-Q for 3 days, and cell functions were assessed by (A) albumin levels measured using an ELISA kit (Sigma), (B) urea levels analyzed using a colorimetric assay kit (BioAssay Systems), and (C) GSH levels measured using a luminescent kit (Promega). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001 as compared to control. Data are means ± SDs (n = 3–8).

Fig. 3

Time dependent changes of albumin and urea levels in culture medium of PHLSs. Levels of secreted albumin and urea were measured using cell culture supernatants of untreated PHLSs or those treated with 0.1 % DMSO for 28 days. The dotted line in the urea plot indicates assay limit of detection. ∗∗p < 0.01 as compared to the control. Data are means ± SDs (n = 3–4).

Fig. 4

LDH leakage from PHLSs treated with 6PPD or 6PPD-Q. PHLSs were treated with 6PPD or 6PPD-Q for 28 days, and LDH activity was measured using culture medium on the specified days. APAP (10 mM) was included as a positive control for LDH activity assay. ∗p < 0.05 as compared to control. Data are means ± SDs (n = 7–24).

Fig. 5

Functional changes of PHLSs in response to 6PPD or 6PPD-Q. PHLSs were treated with 6PPD (A) or 6PPD-Q (B) for 28 days, and then levels of albumin and urea were measured using culture medium on the specified days. APAP (10 mM) was included as a positive control for albumin measurement. Dotted lines in the urea graphs indicate the assay limit of detection. ∗p < 0.05, ∗∗∗∗p < 0.0001 as compared to control. Data are means ± SDs (n = 3–8).

Fig. 6

Effects of 6PPD or 6PPD-Q on cellular GSH, ATP and caspase 3/7 activity in PHLSs. PHLSs were treated with 6PPD or 6PPD-Q for 28 days, and GSH, ATP and caspase 3/7 activity were measured on day 14 (A–C) and day 28 (D–F). All endpoints were assessed using commercial kits from Promega. APAP (10 mM) was included as a positive control. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 as compared to control. Data are means ± SDs (n = 3–6).

Fig. 7

Effects of 6PPD or 6PPD-Q on 10 cytokines secreted from PHLSs. PHLSs were treated with 6PPD or 6PPD-Q for 28 days, and 10 secreted cytokines were measured from cell culture supernatants on day 14 (A) and day 28 (B) using a V-PLEX Plus Proinflammatory Panel 1 Human Kit. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 as compared to control. Data are means ± SDs (n = 3).