Effects of Triclocarban (TCC) on Cytokine and Pulmonary Inflammation

Alexandra Pietrzyk, Nichole Olsen, Melanie Fu, Mithila Venkataraman,

Nancy A. Gee, Christoph F. Vogel, Kent E. Pinkerton and Bill L. Lasley
Center for Health and the Environment, University of California, Davis, CA 95616
ABSTRACT

MATERIALS AND METHODS

CONCLUSIONS

Triclocarban (TCC) is an anti-microbial found in soap

ranging in concentration from 0.1 to 5%. TCC elicits
adverse biological effects in several species. TCC can
increase nuclear receptor-stimulated gene expression
and may play a role in cytokine expression. This study
investigated the effect of TCC on cytokines in vitro
and pulmonary inflammation in vivo, subsequent to
lipopolysaccharide
(LPS)
(an
inflammogen)
stimulation. In vitro, RAW 264.7 cells, a macrophage
cell line, demonstrated that TCC depressed LPSstimulated induction of IL-10 (a anti-inflammatory
cytokine) (p<0.05), while elevating IL-6 (both a proand an anti-inflammatory cytokine) (p<0.05) in a dosedependent fashion. Treatment of RAW 264.7 cells
with budesonide, a corticosteroid, did not alter the
expression of IL-6 or IL-10. In vivo studies were
performed in 7 week old male Sprague Dawley rats
fed a standard diet or a TCC-enriched diet (0.5% w/w)
ad libitum for 10 days. Animals were challenged by a
single intranasal (IN) dose of LPS (5g/animal in 0.2
mL). A subset was given budesonide IN (5mg/kg in
0.2 mL) 2h prior to LPS challenge. Animals were
necropsied 18h post-LPS challenge. Bronchoalveolar
lavage (BAL) was used to determine the degree of
pulmonary inflammation. TCC alone did not
significantly change the inflammatory response due to
LPS induction. Budesonide treatment 2h prior to LPS
challenge led to an overall reduction in inflammation
(measured as neutrophils/ml in BAL), regardless of
diet. A 2-fold reduction in neutrophil numbers was
noted for budesonide treatment following the TCCenriched diet, compared to budesonide treatment with
animals not on the TCC-enriched diet. Although these
in vivo changes did not achieve a level of statistical
significance, this was a consistent trend observed in 5
out of 6 animals in each group treated with
budesonide.
Furthermore,
we
analyzed
the
expression of various CYP450 enzymes and found a
significant increase of CYP1A1 in liver (data not
shown) and lung of TCC-treated animals (Fig 4). We
conclude TCC has the ability to alter cytokine
expression directly in vitro, while in vivo TCC acts by
enhancing the budesonide-directed lowering of the
inflammatory response to LPS challenge.

In vitro cell culture

MDA-kb2 breast cancer cells containing the glucocorticoid receptor (GR) were
incubated with DMSO vehicle (0.1%) or TCC (1M) in the presence of varying
concentrations of cortisol (10-9 to 10-6 M) for 16h and assessed for luciferase
activity.
RAW 264.7 mouse macrophage cells were treated with DMSO (vehicle) or TCC
(1M or 5M) for 4h then stimulated for 12 and 24h. IL-10 and IL-6 expression
was measured at 12h and 24h.

TCC has an effect on inflammatory cells in vitro but

TCC did not have a significant observable effect on
inflammatory cells in vivo.

In vivo animal studies

Sprague Dawley rats
(Male 7 weeks old)

Standard rodent diet for 10

days (powder)

TCC-enriched (0.5% w/w) rodent

diet for 10 days (powder)

Budesonide
(5 mg/kg) IN
(n=6)

Saline IN
(n=6)

Saline IN
(n=6)

Budesonide
(5 mg/kg) IN
(n=6)

LPS challenge (5g) IN

LP08047SF
Necropsy (18h post-LPS)
Bronchoalveolar lavage (BAL)
CYP P450 IA1 tissue analysis

RESULTS
Fig 1

16
Luciferase Activity
(Relative Light Units)

Cortisol + 1uM TCC

Cortisol + 0.1% DMSO

Fig 4

In vivo
Average number of cells/mL of recovered BAL was
higher in animals that received TCC in their diet
although statistical significance was not attained due
to high individual variability.
TCC did not have an effect on the induced
inflammatory response of macrophages and
monocytes.
TCC treatment led to a 12-fold increase of CYP1A1
in lung compared to control animals (Fig 4).
Budesonide caused a further increase of CYP1A1
which was not statistically significant.
A 2-fold decrease in neutrophil number was
observed in BAL recovered from rats that received
budesonide and the TCC-enriched diet when
compared to neutrophil number in BAL recovered
from rats receiving budesonide and the standard diet
(Fig 5).

12

SUMMARY

Because TCC in vitro increases IL-6 and decreases IL10, exposure to TCC may lead to an excessive
inflammatory response which may precipitate
autoimmunity. Furthermore, TCC could pose a health
risk if this pattern were observed in vivo. TCC
amplified the signal transduction of the glucocorticoid
cortisol in vitro and the efficacy of a synthetic
glucocorticoid, budesonide, in vivo, although the latter
did not have statistical significance. Additional studies
need to be performed with larger sample populations.

0
10 -11 10 -10 10 -9 10 -8 10 -7 10 -6 10 -5

Cortisol (M)
(*p<0.05)

Fig 2

*p<0.05 compared to LPS at 12h

**p<0.001 compared to LPS at 12h
***p<0.05 compared to LPS at 24h

Fig 5

INTRODUCTION

**
***

Effect of TCC on neutrophils in BAL

(budesonide-treated/LPS-challenged SD rats)

1.5

Fig 3

***
*p<0.05 compared to LPS at 12h
**p<0.001 compared to LPS at 12h
***p<0.001 compared to LPS at 24h

***

**

ACKNOWLEDGEMENTS
This research was supported by the National Institute
of Environmental Health Sciences (NIEHS) Superfund
Basic Research Program (5P42 ES04699). The
authors gratefully acknowledge the outstanding
research support provided by Janice Peake, Dale
Uyeminami, Imelda Espiritu, Katherine Johnson and
Alexa Pham. This study would not have been
possible without their expertise.

1.0

Cells (x 105)

Triclocarban (TCC; 3,4,4-trichlorocarbanilide) is an

anti-microbial compound used in some bar soaps at
concentrations ranging from 0.1% to 5%. Animal
studies have shown that TCC has the capacity to elicit
adverse biological effects in the development of the
reproductive and endocrine systems, amplifying signal
transduction of many steroid hormones, including
estrogens, androgens and glucocorticoids. TCC may
also increase nuclear receptor-stimulated gene
expression and impact cytokine expression. This
study investigated the effects of TCC on cytokines in
vitro and on pulmonary inflammation in vivo using the
Sprague-Dawley rat model with exposure by TCCenriched diet.

In vitro
TCC significantly enhanced the signal transduction
of cortisol in MDA-kb2 cells containing the GR as
compared to cortisol alone (Fig 1).
TCC depressed the induced inflammatory response
of the anti-inflammatory cytokine IL-10 (Fig 2).
TCC elevated the response of IL-6, which can act
as both a pro-inflammatory and anti-inflammatory
cytokine (Fig 3).

0.5
0.0
-0.5
-1.0

-1.5
-2.0
Standard Diet
(n=6)

TCC Diet
(n=6)