N-Acetyl Cysteine (NAC) Treatment Reduces Mercury-Induced

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N-acetyl cysteine (NAC) treatment reduces mercury-induced

neurotoxicity in the developing rat hippocampus

Anthony Falluel-Morel1,2, Lulu Lin1, Katie Sokolowski1,3, Elizabeth McCandlish4,6, Brian

Buckley4,6, and Emanuel DiCicco-Bloom1,3,5,6

1Department of Neuroscience and Cell Biology, UMDNJ-Robert Wood Johnson Medical School,

Piscataway, New Jersey 08854, USA

2INSERM U982, University of Rouen, 76821 Mont-Saint-Aignan, France

3Joint Graduate Program in Toxicology, Graduate School of Biomedical Sciences, Rutgers/

UMDNJ-Robert Wood Johnson Medical School

4Environmental and Occupational Health Sciences Institute (EOHSI), Rutgers University,

Piscataway, New Jersey 08854, USA

5Department of Pediatrics, UMDNJ-Robert Wood Johnson Medical School, New Brunswick, New

Jersey 08901

6Member, UMDNJ Center for Environmental Exposures and Disease

Abstract

Mercury is an environmental toxicant that can disrupt brain development. However, while

progress has been made in defining its neurotoxic effects, we know far less about available

therapies that can effectively protect brain in exposed individuals. We previously developed an

animal model in which we defined the sequence of events underlying neurotoxicity:

Methylmercury (MeHg) injection in postnatal rat acutely induced inhibition of mitosis and

stimulated apoptosis in the hippocampus, that later resulted in intermediate term deficits in

structure size and cell number. NAC is the N-acetyl derivative of L-cysteine used clinically for

treatment of drug intoxication. Here, based on its known efficacy in promoting MeHg urinary

excretion, we evaluated NAC for protective effects in the developing brain. In immature neurons

and precursors MeHg (3M) induced a >50% decrease in DNA synthesis at 24hr, an effect that

was completely blocked by NAC co-incubation.In vivo, injection of MeHg (5g/gbw) into 7 day-

old rats induced a 22% decrease in DNA synthesis in whole hippocampus and a 4-fold increase in

activated caspase-3 immunoreactive cells at 24hr, and reduced total cell numbers by 13% at 3

weeks. Treatment of MeHg exposed rats with repeated injections of NAC abolished MeHg

toxicity. NAC prevented the reduction in DNA synthesis and the marked increase in caspase-3

immunoreactivity. Moreover, the intermediate term decrease in hippocampal cell number

provoked by MeHg was fully blocked by NAC. Altogether, these results suggest that MeHg

toxicity in the perinatal brain can be ameliorated by using NAC, opening potential avenues for

therapeutic intervention.

Keywords

mercury; hippocampus; N-acetyl cysteine; neurogenesis; programmed cell death

Corresponding Author:Emanuel DiCicco-Bloom, Department of Neuroscience & Cell Biology, Robert Wood Johnson MedicalSchool, 675 Hoes Lane, Room RWJSPH 362, Piscataway, NJ 08854; [email protected]; Tel: 732-235-5381; Fax: 732-235-4990.

NIH Public AccessAuthor ManuscriptJ Neurosci Res. Author manuscript; available in PMC 2013 April 1.

Published in final edited form as:

J Neurosci Res. 2012 April ; 90(4): 743750.

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INTRODUCTION

Methylmercury (MeHg) is an environmental toxicant that poses serious health risks in

humans and especially children, whose brains are still developing and are therefore

particularly vulnerable to exogenous toxicants (Adams et al.2000; Stein et al., 2002;

Spurgeon 2006). MeHg exposure results primarily from the consumption of contaminated

food. MeHg is easily absorbed from the diet into the bloodstream and distributes to all

tissues including the brain. Indeed, MeHg exhibits high mobility in the body, due to itsability to form thiol complexes with small molecules such as the amino acid cysteine

(Clarkson et al.2007). The kinetics of MeHg accumulation in the brain differs from that of

peripheral organs, such as liver or kidney (Burbacher et al., 2005), raising the possibility

that therapeutic interventions may be organ-specific. In the central nervous system, MeHg

interferes with developmental processes, such as neurogenesis and cell survival, as

demonstrated in both humans and animal models (Chang et al., 1977; Lapham et al.1995;

Newland et al.2004; Burke et al.2006; Falluel-Morel et al.2007). Indeed, previous studies

demonstrated that an acute MeHg exposure by subcutaneous injection in 7 day old (P7) rats

induces cell cycle arrest and cell death of neuronal precursors in the dentate gyrus of the

hippocampus (Burke et al.2006; Falluel-Morel et al.2007; Sokolowski et al., 2011). In

humans, it is difficult to estimate the level of exposure in the fetus and in children in

affected areas, and effective treatments for brain toxicity have yet to be defined. The cellular

mechanisms underlying mercury neurotoxicity are not fully understood, although severalstudies now indicate that ROS production plays a central role (Falluel-Morel et al., 2007;

Haase et al., 2011).

N-acetyl cysteine (NAC) is a compound used clinically for the treatment of drug

intoxication, such as acetaminophen, and recent animal studies suggest it is a useful antidote

for peripheral organ metal toxicity (Ballatori et al., 1998). In the adult, NAC reduced body

MeHg levels by promoting rapid urinary excretion. In parallel, NAC can increase the

reserves of the antioxidant glutathione in the body. Like its homologue glutathione, NAC

contains a thiol group that confers antioxidant properties, potentially enhancing cellular

resistance against reactive oxygen species (ROS). However, the utility of NAC as an

antidote in the preweanling animal (


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All animal procedures were approved by the Robert Wood Johnson Medical School

institutional animal care and utilization committee and conformed to NIH Guidelines for

animal use.

Exposure Models

Methylmercury chloride (CH3HgCl) was purchased from Sigma (St Louis, MO). A 1.5 mg/

mL stock solution in 0.1M phosphate buffered saline (PBS) was prepared immediately

before use and dissolved by agitation. Mercury administration and disposal procedures wereapproved by the Environmental and Occupational Health Sciences Institute (EOHSI)

institutional committee. N-acetyl cysteine (NAC) was obtained from Sigma (St. Louis, MO).

A 2 mg/ml stock solution was prepared in PBS.In vitro, stock solutions were dissolved in

the culture medium to reach the final desired concentrations of MeHg (0.1 6 M) and

NAC (3 1,000 M).In vivo, P7 rats were injected subcutaneously (sc) with vehicle or

MeHg (5 g/gbw) in a 100L bolus. Animals were also injected intraperitoneally (IP) with

vehicle or NAC (10 g/gbw) every two hours during an 8 hour period. According to

experimental needs, animals tissues were dissected and processed immediately, or frozen at

80 C until assay.

Cortical Neuron and Precursor Culture

To obtain a relatively homogeneous neuronal population, the dorsolateral cerebral cortexfrom E14.5 rat embryos was separated from the basal ganglia and overlying meninges. At

this stage, immature neurons as well as mitotic precursors exist in the embryo, and after

plating, additional precursors undergo cell cycle exit to begin neuronal differentiation, as

shown previously (Carey et al., 2002). Cells were dissociated mechanically, plated on 0.1

mg/mL poly-D-lysine coated culture dishes, and incubated at 37C with 5% CO2 in defined

media (Lu and DiCicco-Bloom 1997) composed of DMEM and F12 (50:50 v/v; Invitrogen,

Grand Island, NY) and containing penicillin (50U/mL), streptomycin (50 g/mL),

transferrin (100g/mL) (Calbiochem, La Jolla, CA), putrescine (100 M), progesterone (20

nM), selenium (30 nM), glutamine (2 mM), glucose (6 mg/mL), and bovine serum albumin

(10 mg/mL). Unless otherwise noted, components were obtained from Sigma (St. Louis,

MO). Cells (3 105) were added to 24-well plates and were treated with MeHg/drugs 1hr

after plating so that initial adhesion was not disturbed by the treatments.

[3H]-Thymidine Incorporation

Tritiated thymidine (5 Ci/gbw; Amersham Bioscience, UK) was injected sc into animals 2

hrs prior to analysis. DNA synthesis was evaluated using a percent incorporation assay, as

described (Burke et al.2006; Cheng et al.2002; Wagner et al.1999). Frozen tissues were

manually homogenized in distilled water using a 22 gauge needle and syringe. An aliquot

was removed for determination of total isotope uptake into the tissue. In an equal aliquot,

DNA was precipitated with 10% trichloroacetic acid, sedimented by centrifugation, and

washed by resuspension and resedimentation. The final pellet was dissolved and counted

along with the original aliquot in a scintillation spectrophotometer. Since radiolabel

incorporation into DNA depends on the amount of label taken up by the tissue, incorporation

was calculated as the fraction of total tissue uptake. This method assures that experimental

effects do not reflect possible differences in tissue region dissection or individual animal

injection, absorption or blood flow, but rather changes in specific regional DNA synthesis.

DNA synthesis i n vitro

Plated cells were incubated with tritiated thymidine (5 Ci/mL) during the last 2 hrs of total

incubation, detached with a trypsin-EDTA solution, and collected onto filter paper with a

semi-automatic cell harvester (Skatron) (Lu and DiCicco-Bloom 1997). After addition of the

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RESULTS

Effects of NAC on MeHg induced neurotoxici ty in cu lture

To investigate the potential protective effects of NAC on brain cells, we first used an in vitro

model of embryonic cortical neurons and precursors which was previously shown to be

suitable for the study of mercury neurotoxicity (Burke et al.2006; Falluel-Morel et al.2007;

Sokolowski et al., 2011). We treated cortical cultures with various concentrations of MeHg

ranging from 0.1 to 6 M and measured [3H]-Thymidine ([3H]-Thy) incorporation at 24hours. In this model, MeHg induced a concentration-dependent decrease in DNA synthesis

significant at 1.5 M and higher (Fig. 1A). The 3 M MeHg concentration that induced a

90% decrease in [3H]-Thy incorporation was used to investigate the potential effects of

NAC. Several concentrations of NAC were assessed, ranging from 3 to 1,000 M (Fig. 1B).

NAC induced a concentration-dependent protective effect, significant at 30 M and above,

with a complete protection at 300 M. Interestingly, when administered alone, 100300 M

NAC induced a ~30% increase in DNA synthesis. Moreover, the induction of cell death at

24 hrs following MeHg exposure was completely abolished by NAC co-incubation (Fig. 1

C).

As a metabolic precursor, some studies have used culture preincubation with NAC to allow

extended time for cellular uptake and glutathione biosynthesis (Shimizu et al., 2002). Thus

we examined the impact of pretreatment with NAC on its protective effects against MeHgtoxicity (Fig. 1D). Although NAC was highly effective in counteracting MeHg toxicity

when co-administered, it proved entirely ineffective when added to the culture media for a

period of either 4 or 24 hours and then removed prior to mercury exposure.

Effects of NAC on MeHg induced neurotoxici ty in vivo

Because NAC appeared to be a potent and efficacious inhibitor of MeHg-induced

neurotoxicity in cultured cortical neurons and precursors, we investigated its effects further

in a developmental model in vivo. We performed these studies using a paradigm of acute

exposure of perinatal rats to MeHg and assessment of the hippocampus. In this model,

MeHg induces cell cycle arrest and programmed cell death of dentate gyrus neuronal

precursors, leading to intermediate term modification of hippocampal structure and function

(Falluel-Morelet al.

, 2007). In the current experiments, P7 rats were given 5 injections ofNAC (10 g/gbw) with an interval of 2 hours between injections, spanning a total of 8

hours. Concurrent with the second NAC administration, animals received a single injection

of saline or 5 g/gbw MeHg, and [3H]-Thy incorporation was measured 24 hours later (Fig.

2A). This injection paradigm was chosen amongst several administration protocols (see

Discussion below) to ensure sufficient NAC blood levels without producing side effects.

MeHg induced a 20% decrease in DNA synthesis in the total hippocampus, reproducing the

inhibitory effects defined in previous studies (Burke et al., 2005; Falluel-Morel et al., 2007).

However, in the presence of NAC, the negative effects of MeHg were almost completely

blocked. NAC alone had no significant effect. Thus, similar to our in vitrodata, NAC

administration in vivoprevented the inhibitory effects of MeHg on hippocampal DNA

synthesis.

Effects of NAC on mercury levels in the hippocampusThe ability to block MeHg neurotoxic effects in the perinatal rat raised the possibility that

NAC may have altered the metals access to the brain, especially given its ability to promote

MeHg renal uptake and excretion (Koh et al., 2002). To examine this issue, we measured

hippocampal mercury levels 24 hours following exposure (Fig. 2B). The single 5 g/gbw

injection led to the accumulation of ~2,000 ppb Hg in the hippocampus, whereas mercury

levels were almost undetectable in vehicle injected animals. Significantly, in the presence of



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NAC, hippocampal Hg levels were decreased by ~25%, indicating that NAC co-

administration with MeHg reduced mercury accumulation in the hippocampus (P


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its deleterious effects. However, in vivo, the mechanism(s) by which NAC exerts its

neuroprotective effects remains to be elucidated.

Our observations in vivohowever are consistent with the concept that a fraction of

methylmercury binds to cysteine in the blood, forming a compound which can be actively

transported by hepatic and kidney cells into bile and urine (Clarkson et al., 2007). This

excretion is described as a two-step process, with the uptake of MeHg-NAC from blood into

epithelial cells by organic anion transporters, such as Oat1 (Kohet al.

, 2002), followed byactive excretion of the complex into bile or urine by the apical Multidrug resistance-

associated protein-2 (Mrp2/Abcc2) (Madejczyk et al., 2007). Significantly, however, Aremu

and coworkers have shown that this mechanism, which allows efficient, NAC-enhanced

renal excretion of MeHg in adult rat, was not effective in young animals (


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the production of the metabolite S-nitroso-N-acetylcysteine (SNOAC). Similar effects have

been described in humans (Hildebrandt et al.2002). These various data suggest that NAC

dosage regimens may need to be tailored based on both animal species as well as

developmental age.

In addition to insights into preventing MeHg neurotoxicity, the current studies provide

information on the possible mechanism by which the toxicant induces hippocampal

teratogenicity. It is possible that MeHg exposure elicits acute effects on proliferation ofhippocampal neural precursors through blockade of G1/S phase transition and induction of

caspase-dependent apoptosis (Falluel-Morel et al., 2007; Sokolowski et al., 2011), whereas

later effects on hippocampal cell number would be mediated by other pathways. The current

studies however, showing that NAC prevents acute mitotic inhibition as well as later

hippocampal cell deficits, suggest MeHgs main teratogenic mechanism is its acute

inhibition of neural precursor proliferation/survival. While NAC prevented MeHg induced

reduction in hippocampal cell number, future studies will determine whether NAC can also

prevent the spatial learning deficits occurring in this model at puberty (Falluel-Morel et al.

2007).

In conclusion, our studies indicate that injection of NAC is an efficient method to prevent

MeHg-induced toxicity in the perinatal brain in vivoThese studies support the mounting

evidence that NAC may be preferable to the currently available thiol-containing MeHgchelators, meso-2,3- dimercaptosuccinic acid (DMSA) and 2,3-dimercapto-1-

propanesulfonate (DMPS) that unfortunately mobilize and deplete other minerals (especially

divalent cations) that are essential for normal physiologic function (discussed in Aremu et

al., 2007). Our studies in developing hippocampus contrast with other work that suggests

NAC exposure may enhance mercury induced damage by serving as a molecular transporter

(Zalups et al., 2005; Rooney et al., 2007). However, to produce protective effects, low dose

NAC administration was initiated 2 hours prior to MeHg exposure. In preliminary studies,

we did not detect protection when NAC administration was begun 2 hours after MeHg

exposure, suggesting that MeHg was distributed rapidly to sites of injury and/or that proper

NAC dosing for post-MeHg treatment remains to be determined, a possibility under active

investigation. Nonetheless, the current evidence, contrary to previous studies, suggests that

NAC may provide some degree of benefit from brain injury during the perinatal period,

when the sources of mercury exposure are predictable or sustained. Since fish remains agood source of nutrients and oils beneficial for neurodevelopment, potential deleterious

effects of mercury-containing fish consumption may be ameliorated through the use of NAC

or a related compound. Therefore, it is of interest to develop compounds able to bind heavy

metals with high affinity without producing toxic effects by themselves to prevent

developmental neurotoxicity in populations exposed to organomercurials.

Acknowledgments

Grant information:National Institutes of Health (ES11256 to E.D-B., ES05022 to E.D-B., ES07148 to K.S..

NS062591 to K.S., NIH-NIEHS 1R21ES019762 to E.D-B.); UMDNJ Center for Environmental Exposures and

Disease (P30ES005022); Fondation pour la Recherche Mdicale (SPE20051105 to A.F-M.); US Environmental

Protection Agency (R82939101 to E.D-B.).

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Figure 1. NAC prevents MeHg toxic effects on DNA synthesis and cell survival in embryoniccortical cultures

A:Concentration-dependent effects of MeHg on [3H]-Thy incorporation in cortical cultures.

Cells were exposed for 24 hrs to vehicle or 0.1, 1, 1.5, 2, 3 or 6 M MeHg at zero time. [3H]-

Thy was added at 20 hrs and cells were collected at 24 hrs for analysis. MeHg exposure

induced a concentration-dependent reduction in DNA synthesis. B:Concentration-

dependent effect of NAC on MeHg-induced inhibition of DNA synthesis. Cells were

exposed or not to MeHg (3M) and/or NAC (3 1,000M) for 24 hrs. NAC treatment

increased thymidine incorporation at high concentrations (>100M) and induced a dose-

dependent protection against the negative effects of MeHg. C:Protective effect of NAC



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against MeHg-induced cell death. Cells were exposed or not to MeHg (3M) and/or NAC

(300M) for 24 hrs and total numbers were assessed under phase microscopy. NAC

cotreatment completely abolished MeHg-elicited neuronal death. D:Influence of treatment

paradigm on NAC protective effects in vitro. NAC (300M) exerted protective effects only

when it was administered at zero time concurrently with MeHg (3 M). However, when

NAC was added to the culture media either 4 or 24 hours before MeHg and removed prior to

mercury exposure, no protection was measured. Data are expressed as the mean sem of 3

independent experiments for all groups, performed in quadruplicates for every condition.*P


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Figure 2. NAC administration reduces mercury uptake into the hippocampus and preventsinhibition of DNA synthesis in developing P7 rats in vivo

P7 rats were injected with saline or MeHg (5g/gbw) and received 5 repeated injections of

NAC (10g/gbw per injection) over 8 hours with 2 hours intervals between each injection.

NAC exposure was initiated 2 hours before MeHg exposure. A:[3H]-Thy incorporation into

the whole hippocampus was measured 24 hours after MeHg exposure. The inhibitory effect

of MeHg on DNA synthesis was almost completely abolished by NAC. B:ICP-MS

measurement of hippocampal mercury content 24 hours after treatment with NAC and/or

MeHg. Mercury was almost undetectable in the control and NAC treated animals. MeHg sc

injection led to a massive Hg uptake into the hippocampus, which was significantly reduced



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by NAC. Values are expressed as the means sem of 4 independent experiments for all

groups, with 3 animals per group in each experiment (N=12 per group). **, P< 0.01 vs

control; ***, P< 0.001 vscontrol; #, P< 0.05 vsMeHg; ##, P< 0.01 vsMeHg.



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Figure 3. NAC injection prevents acute induction of apoptotic cell death elicited by MeHgexposure in the P7 hippocampal dentate gyrus

A:Detection of activated caspase-3 immunoreactivity in the dentate gyrus following MeHg

and/or NAC exposure. P7 rats were injected with vehicle, 5.0 g/gbw MeHg and/or 510g/

gbw NAC, sacrificed at 24 hrs and processed for immunostaining. Scale bar = 100 m. B:

Quantification revealed that the MeHg induced increase in caspase-3 positive cell number

was completely blocked by NAC. Values are expressed as the means sem per section of 9

sections per animal, 3 animals per group. ***, P< 0.001 vscontrol; ###, P< 0.001 vs

MeHg.



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Figure 4. NAC administration prevents MeHg-induced reduction of hippocampal cell content atP21

A:Measurement of hippocampal DNA content at 2 weeks (P21) after MeHg exposure on

P7. Total DNA was significantly reduced in MeHg treated animals, and NAC injection

prevented this effect. B:Body weights of the animals were measured prior to sacrifice and

no significant differences were observed between animals. Values are expressed as the

means sem of 3 independent experiments, with 3 animals per group in each experiment. *,

P< 0.05 vscontrol ; ###, P< 0.001 vsMeHg.



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N-Acetyl Cysteine (NAC) Treatment Reduces Mercury-Induced