  SEQ CHAPTER \h \r 1 ICF Consulting Review of Fueta et al. (2002)

ICF has been asked to review the journal article, “Hyperexcitability
and changes in activities of Ca2+/calmodulin-dependent kinase II and
mitogen-activated protein kinase in the hippocampus of rats exposed to
1-bromopropane” by Fueta and coworkers (2003).  Details regarding the
study design, results, and major conclusions are detailed in the
following text.  Further, ICF will discuss the relevance of this study
to other studies in rodents and will discuss whether this study allows
for the identification of an alternate NOAEL/LOAEL for the determination
of an Acceptable Exposure Limit for n-propylbromide (i.e.,
1-bromopropane).

Background and Study Purpose

The hippocampus is a section of the brain that in the human is located
within the temporal lobe.  Damage to it or any region that is connected
to it results in the inability to learn new information.  The
hippocampus is divided into four regions:  CA1, CA2, CA3, and CA4.  CA1
and CA3 are very important in hippocampal processes; it is thought that
cells in these regions are involved in the consolidation of long-term
memories, in particular for spatial location.  This learning may occur
via "long-term potentiation" which is an increase in the excitability of
a neuron to a unique synaptic input caused by repeated high-frequency
activity at that input.  Further, learning experiences involving the
hippocampus and associated brain structures induce biochemical changes
that underlie long-term potentiation.  The purpose of this study was to
determine if hyperexcitability in the hippocampus and dentate gyrus
(another brain structure that is part of the hippocampal formation)
caused by 1-bromopropane exposure results in intercellular signaling
changes in the following proteins: Ca2+/calmodulin-dependent kinase II
(CAMKII); mitogen-activated protein kinase (MAPK), and protein kinase C
(PKC).  The first two proteins (CAMKII and MAPK) have been implicated as
important for learning and memory.	

								

Study Design

Male Wistar rats at 7 weeks of age were exposed to target concentrations
of 0 (fresh air) or 700 ppm n-propylbromide (12/group) in whole-body
inhalation chambers for 6 hours/day, 5 days/week for 8 weeks.  Measured
concentrations of the nPB, if analyzed, were not reported. Hippocampal
slices were obtained from the brains of six rats from each exposure
group.  Four or six slices were obtained from each rat and perfused with
an artificial cerebrospinal fluid.  After stabilization of the slices,
microelectrodes were inserted into the CA1 or dentate gyrus. 
Paired-pulse stimulation was conducted, and paired-pulse inhibition or
facilitation was measured (the depression or potentiation in the
responses to the second stimulation relative to those in the first
stimulation).  Presence of active (phosphorylated) enzymes in
homogenized hippocampal tissue from the remaining 6 rats/group was
assayed using immunoblotting techniques with antibodies raised to the
following enzymes:  CaMKII, MAPK, active MAPK, and PKCα.

Study Results

Exposure to 700 ppm nPB for 8 weeks resulted in a significant decrease
in paired-pulse inhibition at 5 ms intervals in the CA1 region and at
both 10 and 20 ms intervals in the dentate gyrus.  No effects were seen
at higher intervals in these two regions.  The study authors indicated
they had published previous results with rats exposed to 1500 ppm nPB;
the results in these animals was not as great as those exposed to the
higher concentration.

n the total amounts of CaMKIIα (by 29%) and CaMKIIβ (by 46%) subunits,
but no increase in the phosphorylated versions of these subunits.  In
fact, phosphorylated CaMKIIβ was significantly decreased (by 22%)
following nPB exposure.  Phosphorylated MAPK was increased by 28%
compared to the control response, with no concomitant change in overall
MAPK concentration.  Total and phosphorylated PKCα levels were
unaffected. 

Discussion

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mpus and dentate gyrus.  However, this increase was not associated with
behavioral affects (according to the study authors) and could not be
causally linked with an increase CaMKII activity.  There may be a link
between the increase in overall CaMKII expression and the
hyperexcitability.  The data suggest that intercellular signaling is
modified, as active levels of MAPK were increased.  These results cannot
be used to provide an alternative AEL as only one exposure concentration
was used (which is higher than the level already associated with other
toxic effects in rodents [400 ppm]) and a shorter exposure duration (8
weeks) was used than the other subchronic studies that have shown
effects (13 weeks).

Reference

Fueta Y, Fukunaga K, Ishidao T, and Hori H. 2002.  Hyperexcitability and
changes in activities of Ca2+/calmodulin-dependent kinase II and
mitogen-activated protein kinase in the hippocampus of rats exposed to
1-bromopropane.  Life Sci 72:521-529.

