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. Author manuscript; available in PMC: 2024 Feb 1.
Published in final edited form as: Arch Toxicol. 2022 Nov 14;97(2):469–493. doi: 10.1007/s00204-022-03413-z

Organotin and organochlorine toxicants activate key translational regulatory proteins in human immune cells

Amanda Ruff 1, Meaghan Lewis 1, Margaret Whalen 2
PMCID: PMC9939003  NIHMSID: NIHMS1868802  PMID: 36372856

Abstract

Environmental contaminant exposures occur due to the widespread use of synthetic chemicals. Tributyltin (TBT), dibutyltin (DBT), and pentachlorophenol (PCP) are each used in a variety of applications, including antifouling paints and stabilizers in certain plastics. Each of these compounds has been found in human blood, as well as other tissues, and they have been shown to stimulate pro-inflammatory cytokine production in human immune cells, Inflammatory cytokines mediate response to injury or infection. However, if their levels are increased in the absence of an appropriate stimulus, chronic inflammation can occur. Chronic inflammation is associated with a number of pathologies including cancer. Stimulation of pro-inflammatory cytokine production by these toxicants is dependent on activation of ERK 1/2 and/or p38 MAPK pathways. MAPK pathways have the capacity to regulate translation by increasing phosphorylation of key translation regulatory proteins. There have been no previous studies examining the effects of TBT, DBT, or PCP on translation. The current study shows that ribosomal protein S6 (S6), eukaryotic initiation factor 4B (eIF4B), and eIF4E are phosphorylated (activated) and/or their total levels are elevated in response to each of these compounds at concentrations found in human blood. Activation/increased levels of translational proteins occurred at concentrations of the compounds that have been shown to elevate pro-inflammatory cytokine production, but where there is no increase in mRNA for those proteins was seen. Compound-stimulated increases in translation appear to be part of the mechanism by which they elevate protein production in immune cells.

Keywords: Tributyltin, Dibutyltin, Pentachlorophenol, eIF4E, eIF4B, S6, Immune cells

Introduction

The organotin compounds, tributyltin (TBT) and dibutyltin (DBT), and the organochlorine, pentachlorophenol (PCP), are synthetic chemicals used in a variety of applications and as such very significantly contaminate the environment. TBT is primarily used as an antifouling paint additive on ships and boat hulls, docks, fishnets, and buoys to discourage the growth of marine organisms (Gipperth 2009; Showalter 2005). TBT has also been used as an additive in several consumer products including siliconized baking parchment (Yamada et al. 1993). It is found at measurable levels in several types of human tissues, including human blood at levels as high as 260 nM (Antizar-Ladislao 2008; Kannan et al. 1995, 1999; Whalen et al. 1999). TBT increases secretion and cellular production of the pro-inflammatory cytokines tumor necrosis factor alpha (TNFα), interferon gamma (IFNγ), interleukin (IL) 1β, and IL-6 in human lymphocytes (Brown et al. 2018a, b; Brown and Whalen 2015; Lawrence et al. 2015, 2018) and these increases are dependent the on p38 and ERK 1/2 MAPK pathway (Brown et al. 2018a; Lawrence et al. 2018). DBT contaminates the environment due to its uses as a stabilizer in polyvinylchloride, PVC plastics (Roper 1992; Yamada et al. 1993). DBT has been found in drinking water and other beverages due to leaching from PVC plastics used during the distribution of drinking water and storage of beverages such as beers and wines (Forsyth et al. 1992; Forsyth and Jay 1997; Sadiki and Williams 1999; Sadiki et al. 1996). Additionally, DBT has been used as a deworming agent in poultry further exposing humans and infiltrating food products (Epstein et al. 1991) and it has been found in human blood at levels as high as 0.3 μM (Kannan et al. 1999; Whalen et al. 1999). DBT, like TBT, can also increase cellular production of IL-1β and IL-6 (Sushak et al. 2020). PCP contaminates the environment due to widespread use in fungicide, herbicide, wood preservative, and in antifouling paints (ATSDR’s Toxicological Profiles 2002; Brown et al. 2005; Cirelli 1978). It has been found in human blood ranging between 0.26 and 5 μM, specifically in individuals that lived in PCP-treated log homes (Cline et al. 1989). An average concentration of 0.15 μM PCP was found in the serum of individuals with no known exposure (Cline et al. 1989; Uhl et al. 1986). As was seen with TBT and DBT, PCP can increase immune-cell production of the pro-inflammatory cytokines, IL-1β and IL-6 (Martin et al. 2019a, b). DBT and PCP, like TBT, require MAPK signaling pathways to produce these increases) (Martin et al. 2019a, b). Thus, TBT, DBT, and PCP all have the potential to cause chronic inflammation in exposed individuals.

Ribosomal protein S6 (S6) is a component of the 40S subunit and is thus essential for translation (Ruvinsky and Meyuhas 2006). Eukaryotic initiation factor 4B (eIF4B) controls protein synthesis by stimulating eIF4F activity through the enhancement of eIF4A RNA helicase activity (Avdulov et al. 2004; Harms et al. 2014; Holz et al. 2005). Importantly, activation (phosphorylation) of these key translational proteins is regulated by MAPK pathways (Roux and Topisirovic 2012). eIF4E binds the 7-methylguanosine-containing cap at the 5-prime terminus of mRNA (5’Cap), and it contributes to the transfer of mRNA to the 40S-S6 ribosomal complex. (Gingras et al. 1999; Morley and Traugh 1989; Pyronnet et al. 1999; Waskiewicz et al. 1997). eIF4E is phosphorylated by MAPKs (Roux and Topisirovic 2012); however, to be fully activated 4E binding protein (4EBP) which inhibits eIF4E must also be phosphorylated by mTOR (Kosciuczuk et al. 2017).

Based on the previous studies indicating that TBT, DBT, and PCP are capable of altering cellular production of pro-inflammatory cytokines, we hypothesize that these compounds are able to activate one or more key translational regulatory proteins, eIF4B, eIF4E, and/or S6, as part of the mechanism by which they increase cytokine production. Here we investigate the ability of TBT, DBT, and PCP at concentrations that have previously been shown to activate pro-inflammatory cytokine production in immune cells to activate one or more of these key translational-regulatory protein.

Materials and methods

Preparation of peripheral blood mononuclear cells (PBMCs)

PBMCs were isolated from Leukocyte filters (PALL-RCPL or RC2D) obtained from the Red Cross Blood Bank Facility in Nashville, TN (Meyer et al. 2005). Leukocytes were retrieved from the filters by back-flushing them with an elution medium (sterile phosphate-buffered saline (PBS) containing 5 mM disodium EDTA and 2.5% [w/v] sucrose, pH 7.2) and collecting the eluent. The eluent was layered onto Lymphosep—Lymphocyte Separation Medium (1.077 g/mL) (Fisher Scientific, Pittsburg, PA), and centrifuged at 1200g for 30 min. Mononuclear cells were collected and washed with PBS by centrifuging at 500g, 10 min. Following washing, the cells were layered on bovine calf serum for platelet removal. The cells were then suspended in complete medium which consists of RPMI-1640 supplemented with 10% heat-inactivated BCS, 2 mM L-glutamine and 50 U penicillin G with 50 μg of streptomycin/mL.

Chemical preparations

TBT, DBT, and PCP were purchased from Sigma-Aldrich, (St. Louis, MO) and Thermo Fisher Scientific (Waltham, MA). TBT chloride is a neat standard, dissolved initially in deionized water to give a 1 mM solution. DBT dichloride (a solid) was dissolved in dimethylsulfoxide (DMSO) to give a 0.685 M solution. A stock solution of PCP was made by dissolving solid PCP in DMSO to give a 100 mM stock solution. Desired concentrations of each compound were prepared by dilution of the stock solution for each compound into cell culture media. Appropriate DMSO controls were used for DBT and PCP.

Cell treatment and lysis

PBMCs were treated with TBT at concentrations of 0, 2.5, 5, 10, 25, 50, 100, 200 nM. Immune cells were treated with DBT and PCP at concentration of 0, 0.05, 0.1, 0.25, 0.5, 1, 2.5, 5 μM Lengths of exposure were 10 min (min), 1 h (h), 6 h and 24 h. Following the treatments, the cells were centrifuged, supernatants collected, and the cell pellets were lysed using 130–150 μL of lysis buffer (Active Motif, Carlsbad, CA or Fisher Scientific) per 3–4 million cells. Cell lysates were immediately stored at − 80 °C until the sample was prepared to be run on SDS-PAGE.

Western blot

Cell lysates were run on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride (PVDF) membrane. The membrane was then immunoblotted with specific primary antibodies: eIF4E, eIF4B, S6, phosphorylated eIF4E, phosphorylated eIF4B (S406 and S422), phosphorylated S6, (Cell Signaling Technology, Danvers, MA), and β-actin (Sigma). Antibodies were visualized using the ECL chemilu-minescent detection system (Fisher Scientific) using a UVP/Analytik-Jena Imager (Analytik Jena, Upland, CA). Density of each protein band was determined by densitometric analysis using the VisionWorks image analysis software (Analytik Jena). Differences in protein expression are determined compared to the control. β-Actin levels were determined for each treatment to monitor protein loading. The density of each protein band was normalized to β-actin to correct for minor differences in protein loading among the lanes.

Statistical analysis

The analysis of variance (ANOVA) and Student’s t test were used to analyze statistical significance. A significant ANOVA was followed by pair wise analysis of control versus exposed data using the Student’s t test, a p < 0.05 considered statistically significant.

Results

Effects of 10 min, 1 h, 6 h, and 24 h exposures to TBT on phosphorylated S6 (P-S6) and total S6 in human immune cells

Figure 1 details the effects of varying lengths of exposure to TBT at concentrations of 2.5–200 nM on the levels of P-S6 and total S6 in human PBMCs (each length of exposure was examined in cells from four individual donors). There were no statistically significant increases seen in P-S6 or total S6 at any concentration of TBT after 10 min, 1 h or 6 h exposures (Fig. 1) (bar graphs show average change compared to control across 4 separate experiments (using cells from different donors) representative western blots for each time point are also shown below the bar graphs). While cells from some donors showed increases in total S6 these were inconsistent across cells from different donors leading to a lack of statistical significance. After a 24 h exposure to 50 nM TBT a 1.3-fold increase in total S6 (p < 0.05) was seen (Fig. 1D). No other TBT exposures caused statistically significant increases in either P-S6 or total S6 after 24 h.

Fig. 1.

Fig. 1

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Effect of 10 min, 1 h, 6 h, and 24 h exposures to TBT on ribosomal protein S6 in human immune cells. A 10 min exposures to control, 2.5, 5,10, 25, 50, 100, and 200 nM TBT on levels of P-S6 and total S6. B 1 h exposure to TBT. C 6 h exposure to TBT. D 24 h exposure to TBT. Bar graphs show the average fold change in either the phosphorylated form of S6, P-S6 or total S6 ± SD at each concentration of TBT. The values are the average of the changes ± SD from experiments using cells from 4 distinct donors. An asterisk indicates a significant increase (p < 0.05). Data from a representative western blot is also shown for each time point

Effects of 10 min, 1 h, 6 h, and 24 h exposures to TBT on phosphorylated eIF4B (P-eIF4B) and total eIF4B in human immune cells

Changes in P-eIF4B and total eIF4B following 10 min, 1 h, 6 h, and 24 h exposures to TBT are shown in Fig. 2 (average change compared to control across 4 separate experiments (using cells from different donors)). Two phosphorylation sites on eIF4B were monitored, S422 and S406. After 10 min exposures to 10 and 25 nM TBT, there were significant increases (p < 0.05) in P-eIF4B (S406) of 1.5-fold. Additionally, a statistically significant increase in total eIF4B levels of 1.4-fold (p < 0.05) was observed in samples exposed to 2.5 nM of TBT (Fig. 2A). Following a 1 h exposure to TBT there was a statistically significant 1.5-fold increase in activated eIF4B levels (phosphorylated at Serine 406) in immune cells treated with 2.5 and 10 nM TBT (p < 0.05, Fig. 2B). No statistically significant increases in activated or total eIF4B were seen after 6 h exposure to TBT at any concentration (Fig. 2C). However, after 24 h exposures to TBT (Fig. 2D) there were statistically significant increases in the P-eIF4B (S422) of 1.4-fold (50 nM TBT) and P-eIF4B (S406) of 1.8-fold at the 200 nM TBT exposure. Total eIF4B was increased at the 2.5 and 5 nM TBT exposures after 24 h with an average increase of 1.6 and 1.5-fold (p < 0.05), respectively.

Fig. 2.

Fig. 2

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Effect of 10 min, 1 h, 6 h, and 24 h exposures to TBT on eukaryotic initiation factor 4B (eIF4B) in human immune cells. A 10 min exposures to control, 2.5, 5, 10, 25, 50, 100, and 200 nM TBT on levels of P-eIF4B (S422), P-eIF4B (S406) and total eIF4B. B 1 h exposure to TBT. C 6 h exposure to TBT. D 24 h exposure to TBT. Bar graphs show the average fold change in either the phosphorylated form of eIF4B (S422), eIF4B (S406) or total eIF4B ± SD at each concentration of TBT. The values are the average of the changes from experiments using cells from 4 distinct donors. An asterisk indicates a significant increase (p < 0.05). Data from a representative western blot is also shown for each time point

Effects of 10 min 1 h, 6 h, and 24 h exposures to TBT on phosphorylated eIF4E (P-eIF4E) and total eIF4E in human immune cells

There were no statistically significant increases in either P-eIF4E or total eIF4E after wither 10 min or 1 h exposures to TBT at any concentration (Fig. 3A, B). However, after 6 h there were increases in P-eIF4E of 1.6, 1.7, and 1.5-fold (P < 0.05) at the 50, 100, and 200 nM concentrations of TBT, respectively (Fig. 3C). A 24 h exposure resulted in an increase of 1.2-fold in total eIF4E (p < 0.05) at 100 nM TBT (Fig. 3D).

Fig. 3.

Fig. 3

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Effect of 10 min, 1 h, 6 h, and 24 h exposures to TBT on eukaryotic initiation factor 4E (eIF4E) in human immune cells. A 10 min exposures to control, 2.5, 5, 10, 25, 50, 100, and 200 nM TBT on levels of P-eIF4E and total eIF4E. B 1 h exposure to TBT. C 6 h exposure to TBT. D 24 h exposure to TBT. Bar graphs show the average fold change in either the phosphorylated form of eIF4E or total eIF4E ± SD at each concentration of TBT. The values are the average of the changes from experiments using cells from 4 distinct donors. An asterisk indicates a significant increase (p < 0.05). Data from a representative western blot are also shown for each time point

Effects of 10 min, 1 h, 6 h, and 24 h exposures to DBT on phosphorylated S6 (P-S6) and total S6 in human immune cells

A 10-min exposure to DBT at concentrations of 5–0.05 μM caused no statistically significant increases in on the levels of phosphorylated (activated) or total S6 in human PBMCs from four individual donors (Fig. 4A). After 1 h, DBT at 0.05, 0.1, 1, and 5 μM increased P-S6 by 1.4, 1.3, 1.9. and 1.8-fold (p < 0.05), respectively (Fig. 4B). Following 6 h of exposure to 2.5 and 5 μM DBT there were again increases in P-S6 of 2.5 and 3.1-fold (Fig. 4C). A 24 h exposure to the lowest concentration of DBT, 0.05 and 0.1 μM increased total S6 by 1.2-fold (p < 0.05) (Fig. 4D).

Fig. 4.

Fig. 4

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Effect of 10 min, 1 h, 6 h, and 24 h exposures to DBT on ribosomal protein S6 in human immune cells. A 10 min exposures to control, 0.05, 0.1, 0.25, 0.5, 1, 2.5 and 5 μM DBT on levels of P-S6 and total S6. B 1 h exposure to DBT. C 6 h exposure to DBT. D 24 h exposure to DBT. Bar graphs show the average fold change in either the phosphorylated form of S6, P-S6 or total S6 ± SD at each concentration of DBT. The values are the average of the changes ± SD from experiments using cells from 4 distinct donors. An asterisk indicates a significant increase (p < 0.050). Data from a representative western blot are also shown for each time point

Effects of 10 min, 1 h, 6 h, and 24 h exposures to DBT on phosphorylated eIF4B (P-eIF4B) and total eIF4B in human immune cells

P-eIF4B (S406) was increased at both 0.05 and 0.1 μM DBT after a 10 min exposure (Fig. 5A). The increase was 1.7-fold at 0.05 μM DBT and 2.8-fold at 0.1 μM DBT. Additionally, there was a statistically significant increase of 3.6-fold in total eIF4B in cells treated with 0.1 μM DBT (Fig. 5A). P-eIF4B (S406) levels were increased after a 1 h exposure to 0.1 (1.5-fold), 2.5 (2.4-fold) and 5 (2.5-fold) μM DBT (p < 0.05) (Fig. 5B). Additionally, 0.1 μM DBT caused an increase of 1.9-fold in total eIF4B after 1 h (p < 0.05) (Fig. 5B). DBT at most concentrations was able to significantly increase the levels of P-eIF4B phosphorylated at S406 after a 6 h length of exposure (Fig. 5C). Increases were of 1.7, 2.5, 2.6, 2.3, and 2.6-fold (p < 0.05) were seen at 0.1, 0.25, 0.5, 2.5, and 5 μM DBT, respectively.

Fig. 5.

Fig. 5

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Effect of 10 min, 1 h, 6 h, and 24 h exposures to DBT on eukaryotic initiation factor 4B (eIF4B) in human immune cells. A 10 min exposures to control, 0.05, 0.1, 0.25, 0.5, 1, 2.5 and 5 μM DBT on levels of P-eIF4B (S422), P-eIF4B (S406) and total eIF4B. B 1 h exposure to DBT. C 6 h exposure to DBT. D 24 h exposure to DBT. Bar graphs show the average fold change in either the phosphorylated form of eIF4B (S422), eIF4B (S406) or total eIF4B ± SD at each concentration of DBT. The values are the average of the changes from experiments using cells from 4 distinct donors. An asterisk indicates a significant increase (p < 0.05). Data from a representative western blot are also shown for each time point

A statistically significant increase in total eIF4B levels was also observed in cells treated with 0.5, 1, 2.5, and 5 μM DBT after 6 h. The increases were between 2 and 2.7-fold (Fig. 5C).

No statically significant changes in P-eIF4B or total eIF4B were seen after 24 h exposures to DBT (Fig. 5D).

Effects of 10 min, 1 h, 6 h, and 24 h exposures to DBT on phosphorylated eIF4E (P-eIF4E) and total eIF4E in human immune cells

Exposure of immune cells (from a minimum of 4 different donors) to DBT resulted in no statistically significant changes in either P-eIF4E or total eIF4E at any length of exposure with the exception of a small but significant change (1.2-fold) in total eIF4E at 2.5 μM DBT after a 1 h exposure (Fig. 6B).

Fig. 6.

Fig. 6

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Effect of 10 min, 1 h, 6 h, and 24 h exposures to DBT on eukaryotic initiation factor 4E (eIF4E) in human immune cells. A 10 min exposures to control, 0.05, 0.1, 0.25, 0.5, 1, 2.5 and 5 μM DBT on levels of P-eIF4E and total eIF4E. B 1 h exposure to DBT. C 6 h exposure to DBT. D 24 h exposure to DBT. Bar graphs show the average fold change in either the phosphorylated form of eIF4E or total eIF4E ± SD at each concentration of DBT. The values are the average of the changes from experiments using cells from 4 distinct donors. An asterisk indicates a significant increase (p < 0.05). Data from a representative western blot are also shown for each time point

Effects of 10 min, 1 h, 6 h, and 24 h exposures to PCP on phosphorylated S6 (P-S6) and total S6 in human immune cells

PBMCs exposed to PCP for 10 min and 1 h showed no statistically significant increases in either P-S6 or total S6 (Fig. 7A, B). However, after 6 h there were increases in P-S6 at 1 and 5 μM PCP of 1.2 and 1.5-fold (p < 0.05) (Fig. 7C). At 24 h there was an increase in total S6 at only one concentration of PCP, 0.25 μM of 1.2-fold (p < 0.05).

Fig. 7.

Fig. 7

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Effect of 10 min, 1 h, 6 h, and 24 h exposures to PCP on ribosomal protein S6 in human immune cells. A 10 min exposures to control, 0.05, 0.1, 0.25, 0.5, 1, 2.5 and 5 μM PCP on levels of P-S6 and total S6. B 1 h exposure to PCP. C 6 h exposure to PCP. D 24 h exposure to PCP. Bar graphs show the average fold change in either the phosphorylated form of S6, P-S6 or total S6 ± SD at each concentration of PCP. The values are the average of the changes ± SD from experiments using cells from 4 distinct donors. An asterisk indicates a significant increase (p < 0.05). Data from a representative western blot are also shown for each time point

shown in Fig. 7B.

Effects of 10 min, 1 h, 6 h, and 24 h exposures to PCP on phosphorylated eIF4B (P-eIF4B) and total eIF4B in human immune cells

Exposure of PBMCs to PCP for 10 min resulted in very little change in P-eIF4B or total eIF4B. There was a small but statistically significant increase seen in P-eIF4B (S422) of 1.2-fold at 2.5 μM PCP (Fig. 8A). There were no significant increases in either P-eIF4B or total eIF4B when cells were exposed to PCP for 1 h (Fig. 8B). However, when cells were exposed to PCP for 6 h there were increases seen in P-eIF4B (S406) at 2.5 and 5 μM PCP of 1.3 and 1.8-fold (p < 0.05) and of total eIF4B of 2.1-fold at 5 μM PCP (Fig. 8C). While a 24 h exposure to PCP resulted in increases in P-eIF4B (S406) at 5 μM PCP and increases in total eIF4B at both 2.5 and 5 μM PCP (p < 0.05) (Fig. 8D). There was also an increase in P-eIF4B (S422) at 0.25 μM PCP of 1.3-fold (Fig. 8D).

Fig. 8.

Fig. 8

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Effect of 10 min, 1 h, 6 h, and 24 h exposures to PCP on eukaryotic initiation factor 4B (eIF4B) in human immune cells. A 10 min exposures to control, 0.05, 0.1, 0.25, 0.5, 1, 2.5 and 5 μM PCP on levels of P-eIF4B (S422), P-eIF4B (S406) and total eIF4B. B 1 h exposure to PCP. C 6 h exposure to PCP. D 24 h exposure to PCP. Bar graphs show the average fold change in either the phosphorylated form of eIF4B (S422), eIF4B (S406) or total eIF4B ± SD at each concentration of PCP. The values are the average of the changes from experiments using cells from 4 distinct donors. An asterisk indicates a significant increase (p < 0.05). Data from a representative western blot are also shown for each time point

Effects of 10 min, 1 h, 6 h, and 24 h exposures to PCP on phosphorylated eIF4E (P-eIF4E) and total eIF4E

None of the concentrations of PCP at any of the lengths of exposure resulted in statistically significant increases in either P-eIF4E or total eIF4E. These results are shown in Fig. 9A–D.

Fig. 9.

Fig. 9

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Effect of 10 min, 1 h, 6 h, and 24 h exposures to PCP on eukaryotic initiation factor 4E (eIF4E) in human immune cells. A 10 min exposures to control, 0.05, 0.1, 0.25, 0.5, 1, 2.5 and 5 μM PCP on levels of P-eIF4E and total eIF4E. B 1 h exposure to PCP. C 6 h exposure to PCP. D 24 h exposure to PCP. Bar graphs show the average fold change in either the phosphorylated form of eIF4E or total eIF4E ± SD at each concentration of PCP. The values are the average of the changes from experiments using cells from 4 distinct donors. Data from a representative western blot are also shown for each time point

Discussion

TBT, DBT, and PCP are used in a variety of applications and due to these uses, they very significantly contaminate the environment and have made their way into human tissues, including blood, at measurable levels (Antizar-Ladislao 2008; Cline et al. 1989; Kannan et al. 1995, 1999; Whalen et al. 1999). Previous studies have shown that each of these compounds (at levels that have been found in human blood) is able to increase the cellular production of the pro-inflammatory cytokines IL-1β and IL-6 (Lawrence et al. 2018; Martin et al. 2019a, b; Sushak et al. 2020). These same studies demonstrated that compound-induced increases in these cytokines were at least in part dependent on the p38 and/or ERK1/2 MAPKs. Both TBT and DBT were able to increase the mRNA for these cytokines within 24 h of exposure but only at some concentrations. There were certain concentrations of the compounds that caused increased protein production but did not increase mRNA levels (Brown et al. 2018a; Sushak et al. 2020). PCP did not consistently increase mRNA levels at any of the concentrations that were able to increase cytokine production (Martin et al. 2019a, b). Activation of the translational process can occur in the absence of increased mRNA for rapid responses to external stimuli (Holland et al. 2004; Mamane et al. 2006). Thus, it is possible that the increases in production of these cytokines in the absence of increased mRNA are due to activation of the translational process. The current study investigates the effects of each of these toxicants on the activation state and overall levels of 3 key translational factors, S6, eIF4B, and eIF4E in immune cells. There have been no previous studies examining the effects of any of these compounds on the activation state/levels of translational regulators.

Each of the compounds, TBT, DBT, and PCP, increased the activation state and/or levels of one or more of the translational proteins. Starting as early as 10 min, TBT was able to statistically significantly increase P-eIF4B at the serine 406 site (S406) at lower concentrations. This same pattern maintained after 1 h of exposure. After longer exposures (6 h and 24 h) there were increases in P-eIF4E and P-eIF4B (both at S406 and S422) as well as for total S6, eIF4B, and eIF4E, which were seen over a wide range of TBT concentration. These data indicate that TBT is able to either activate and/or increase the levels of each of these critical translational activators, at concentrations which have been seen in human blood samples (Kannan et al. 1999; Whalen et al. 1999). DBT increased P-S6 and P-eIF4B (S406) as well as total eIF4B over a wide range of concentrations after 1 h of exposure and these increases maintained after 6 h of exposure. By 24 h most of the increases has subsided except for those seen in total S6 at the two lowest DBT concentrations (0.05 and 0.1 μM, which are in the range seen in human blood samples (Kannan et al. 1999; Whalen et al. 1999)). PCP was able to increase translation regulatory protein activation and/or levels most significantly after 6 h and 24 h and those increases were seen predominantly at the 1, 2,5, and 5 μM exposures, which are concentrations seen in human serum samples (Cline et al. 1989). PCP’s major effect was on increasing eIF4B phosphorylation at S406 as well as elevating total eIF4B.

Cellular production of the inflammatory cytokines IL-1β and IL6 was increased at every TBT concentration examined (2.5–200 nM) after a 24 h exposure (Brown et al. 2018a). However, the only concentration that showed consistent increases in the mRNA for these proteins was 100 nM (Brown et al. 2018a). Thus, it appeared that increases in these proteins stimulated by TBT might be due to increases in translation. The current study indicates that there were statistically significant increases in the activation state and/or overall levels of 1 or more of the key translational proteins beginning after a 6 h exposure to TBT at every concentration tested. While increases in mRNA level may contribute sub-stantially to the increased levels of IL-1β and IL-6 at some concentrations and in some donors. The increase in translational protein activity/levels could account for increases seen at all concentrations. DBT also increased immune cell production of these two inflammatory proteins. IL-1β was consistently increased at 0.05 and 0.1 μM DBT after 24 h and IL-6 was increased at 0.1, 0.25, and 0.5 μM DBT at 24 h. There were consistent increases in the mRNA for IL-1β at 0.1 μM DBT but not at 0.05 μM by 24 h (Sushak et al. 2020). However, the mRNA for IL-6 was increased at each of the concentrations that showed consistent increased in the protein (Sushak et al. 2020). Thus, increases in mRNA could explain the increased levels of protein in most cases. However, the current study showed that there were statisitcally significant increases in the activation state/level of S6 and eIF4B beginning at 1 h and 6 h and a continued increase in total S6 at 24 h at the 0.05 and 0.1 μM DBT exposures. Thus, the increase in IL-1β production at 0.05 μM DBT exposures may be accounted for by increased translation rather than and increase in the mRNA. Exposure to PCP was also able to increase immune cell production of IL-1β and IL-6 (Martin et al. 2019a, b). The increases in IL-1β were consistently seen at most exposures (5, 2.5, 1, 0.25, and 0.1 μM) (Martin et al. 2019a, b). As was seen with TBT there were no consistent increases in the mRNA for IL-1β at those concentrations after 2 h, 6 h, or 24 h exposures (Martin et al. 2019a, b). However, here we show that there were increases in either the activated form and/or total protein for S6 and eIF4B at all of the concentrations that showed increased IL-1β at either 6 h or 24 h, with the exception of 0.1 μM.

Previous studies have shown that the ability of TBT, DBT, and PCP to increase immune cell production of IL-1β and IL-6 is at least in part dependent on activation of MAPKs (Brown et al. 2018a; Martin et al. 2019a, b; Sushak et al. 2020). As mentioned earlier, activation of S6 and eIF4B is regulated by MAPK pathways (Roux and Topisirovic 2012) and these 2 translation regulators are required for both 5’cap-dependent and -independent translation. eIF4E binds the 7-methylguanosine-containing cap and thus is required for Cap-dependent translation (Gingras et al. 1999; Morley and Traugh 1989; Pyronnet et al. 1999; Waskiewicz et al. 1997). eIF4E is also phosphorylated by MAPKs (Roux and Topisirovic 2012) as part of its activation, However, another necessary component for eIF4E activation is phosphorylation of 4E binding protein (4EBP) by mTOR (Kosciuczuk et al. 2017). In the current study, we saw predominantly activating phosphorylation of S6 and eIF4B, which can be achieved by activation of MAPKs, and much less activation of eIF4E. Cellular stress has been shown to inhibit cap-dependent translation while stimulating cap-independent translation by assembly of the translational machinery at internal ribosome entry sites (IRES) (Godet et al. 2019). As each of these compounds inflicts significant stress on the cell, it might be anticipated that translation would be driven by IRES (cap-independent). The current results are consistent with that interpretation, as well as the previously demonstrated role of MAPKs in toxicant-stimulated elevation of IL-1β and IL-6. S6 and eIF4B, which are both required in cap-independent translation, are activated/increased to a greater extent than eIF4E when cells are treated with each of the compounds. Additionally, the current study suggests that IL-1β and IL-6 may be proteins whose mRNAs have IRES.

It is important to understand how compounds that contaminate the environment and enter the human system are able to cause the unwarranted increases in pro-inflammatory cytokines such as IL-1β and IL-6 (Brown et al. 2018a; Martin et al. 2019a, b). Inappropriate elevation of these cytokines as is seen with exposures to TBT, DBT, and PCP, could lead to chronic inflammation. Chronic inflammation is an underlying cause of and/or accompanies a number of diseases such as atherosclerosis, diabetes, rheumatoid arthritis, and cancer (Agostini et al. 2004; Dinarello 2011; Duncan et al. 2003; Gabay 2006).

In summary, the data presented in this study show that each of the compounds were able to increase the activation state and/or levels of one or more key translation regulatory protein in human immune cells within 24 h of exposure. TBT stimulated activation of translational proteins within 10 min, while DBT stimulation occurred as early as 1 h following exposure, and PCP was predominantly effective at 6 and 24 h. The activation of translation was seen at concentrations of each of the compounds that caused increases in pro-inflammatory cytokine production in immune cells. mRNA for these cytokines is not consistently increased at concentrations of the compounds that lead to increases in their cellular production. Increases in translational activation shown here are part of the consequence of immune cell exposure to levels of theses toxicants that are within the range found in human blood. Toxicant-stimulated increases in translation appear to be part of the mechanism by which they elevate protein production in human immune cells.

Acknowledgements

Supported by Grant U54CA163066 from the National Institutes of Health.

Footnotes

Conflict of interest The authors declare that they have no conflict of interest.

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