Effects of radiation exposure and dietary iron on liver metabolic gene expression.
V. E. Wotring, Ph.D.
Pharmacology Discipline, NASA Johnson Space Center
Universities Space Research Association, Houston TX
W130
1180.7
INTRODUCTION
RESULTS
Liver function, especially the rate of its metabolic enzyme
activities, determines the concentration of circulating drugs as
well as the duration of their efficacy. Most pharmaceuticals are
metabolized by the liver, and clinically-used medication doses
are given with normal liver function in mind. A drug overdose
can result in the case of a liver that is damaged and removing
pharmaceuticals from the circulation at a rate slower than
normal. Alternatively, if liver function is elevated and
removing drugs from the system more quickly than usual, it
would be as if too little drug had been given for effective
treatment. Because of the importance of the liver in drug
metabolism, we want to understand any effects of spaceflight
on the enzymes of the liver.
In a preliminary analysis, 13 of 25 genes demonstrated no significant expression changes with any treatment condition,
determined by a relative expression change of at least 2-fold with p <0.05. Selected results are shown below.
(relative to control)
% expression change
300
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*
100
Cyp2e1
Cyp19a1
Cyp17a1
*
Cyp1a2
200
(relative to control)
% expression change
*
* *
*
100
0
-100
-200
-300
*
*
Mt2a
(relative to control)
Mthfr
Mt3
1400
% expression change
NFκB
*
The ABC transporter genes tested exhibited very different
responses. Abcb1b (MDR efflux pump, also involved in
lipid and steroid transport) showed significant expression
increases upon radiation treatment, but high Fe had no
effect. Note the difference in scale from the other figures;
these expression changes are roughly 13-fold higher than
controls. Abcb4 showed no changes under any of the
treatments.
200 50
Alox15
Alox5
Abcb1b
Abcb4
200
*
150
*
100
*
0
Fth1
Ftl
Although this was a preliminary study and the gene expression
results have yet to be examined at the protein level, some
interesting trends are evident.
It has previously been shown that gamma radiation causes
physiological oxidation (Ding, et.al., 2005). Some of the
affected genes in this study are involved in reduction or
removal of oxidized compounds. Increased in expression of
Gpx is likely explained by higher physiological oxidation. Mt2
(metallothionein) is usually thought to remove heavy metals
from the body, but may also play a role in inflammation and
oxygen free radical regulation (Sato et al., 2002). Expression of
this gene is regulated by redox state (which can be affected by
radiation exposure) in addition to metal concentrations.
Increases in metallothionein expression have previously been
reported in livers of fish exposed to 75 mGy γ radiation (Olsvik
et al., 2010).
An 11-to 13-fold increase in expression of transporter Abcb1b
was seen in both radiation-treated groups. Another member of
the same gene family Abcb4 was not affected by any treatment.
Both the magnitude and specificity of this result merit
additional examination. This result also carries the potential for
clinically-relevant alterations in pharmacokinetics of
administered medications, as does the increased expression of
Cyp2e1.
It seems likely that radiation exposure triggers a variety of
homeostatic mechanisms, which could include alterations of
gene expression. Whether high dietary iron exacerbates
oxidative stress is not clear at the gene expression level in the
liver; future examinations of protein expression should inform
this question. Better understanding of these pathways could aid
in development of new countermeasures to ameliorate damage
or maintain health of cells and tissues during spaceflight.
REFERENCES
Ding, LH, M Shingyoji, F Chen, JJ Hwang, S Burma, C Lee, JF Cheng, and DJ
Chen (2005) Gene expression profiles of normal human fibroblasts after exposure to
ionizing radiation: a comparative study of low and high doses. Radiat Res 164(1):
17-26.
Olsvik, PA, LS Heier, BO Rosseland, HC Teien, and B Salbu (2010) Effects of
combined gamma-irradiation and metal (Al+Cd) exposures in Atlantic salmon
(Salmo salar L.). J Environ Radioact 101(3): 230-6.
Sato, M, and M Kondoh (2002) Recent studies on metallothionein: protection
against toxicity of heavy metals and oxygen free radicals. Tohoku J Exp Med
196(1): 9-22.
Iron storage genes were differentially affected by
treatments
The light chain of iron storage protein ferritin (Ftl) showed
elevated expression in the high Fe group and the Rad group, with
a trend toward higher expression in the group that received both
treatments. A very similar pattern was seen in gene expression of
the ferritin heavy chain, (Fth1) but expression changes were
much smaller in magnitude.
*
50
-50
Metallothioneins (Mt) are involved in regulation of metals in
the body, and may act as antioxidants. Expression of the
metallothionein 2 gene decreased by almost 4-fold in the
radiation group, but was unchanged by high Fe. Both
treatments together resulted in a smaller, and insignificant
decrease in expression. Methylenetetrahydrofolate reductase
(Mthfr), involved in amino acid and antioxidant synthesis,
expression was significantly increased by both treatments
together. Expression of Gpx1 (glutathione peroxidase), a very
important antioxidant gene, showed modest but significant
increases in both radiation treatments, but was unaffected by the
high Fe. Mt3 and NFΚB expression were not significantly
affected by any treatment.
Drug transporter genes were differentially affected
by treatments
100
400
-200
Gpx1
Antioxidant genes were differentially affected by
treatments
*
1200
250
1000
200
800
150
600
0 0
(relative to control)
After treatments were completed, animals were anesthetized and
sacrificed. Livers were removed immediately and flash-frozen in
liquid nitrogen. Tissue was homogenized, RNA extracted
(Absolutely RNA, Agilent), purified and quality-tested (Agilent
2100 Bioanalyzer). Complementary DNA was prepared from
high-quality RNA samples (RIN > 8; RT2 First Strand,
Qiagen/SABiosciences), and used to run RT-qPCR screening
arrays for DNA Repair and Drug Metabolism (RT2 Profiler
Arrays, Qiagen/SABiosciences). There were 8 animals in each
treatment group, and one liver sample from each was analyzed
with qRT-PCR. The data presented are means of the 8 samples.
Significance at p< 0.05 determined by Student’s t-test and
compared to control diet sham irradiated animals is indicated by
*. Data were normalized to a set of reference genes whose
expression was not significantly altered by any treatment (Snn,
Ccnh, Atxn). All data are expressed as % change in gene
expression normalized to reference gene expression and
compared to the sham exposed normal diet group.
The cytochrome p450 genes tested exhibited a range of
responses. Cyp2e1 (metabolizes anesthetics and acetaminophen)
was doubled in the animals that received both treatments.
Expression of Cyp19a1 (converts androgens in to estrogen) was
highly variable among the samples in each group; although
expression trended higher in radiation treatment groups, the
changes were not significant. Cyp17a1 (produces cholesterol and
steroid hormones) and Cyp1a2 (metabolizes many administered
medications) showed no significant changes.
-100
% expression change
All procedures were approved by the JSC Animal Care and Use
Committee. Male Sprague-Dawley rats were divided into
control and 3 treatment groups of 8 animals each: high iron diet,
radiation treated, and both high iron diet + radiation treatment.
“Chronic” radiation treatment: The irradiated animals were
exposed to gamma radiation (0.375 Gy/exposure) from a Cs-137
source (energy = 662 KeV) every other day for 16 days (8 doses)
for a total whole body dose of 3 Gy. Non-irradiated animals
(sham) were handled similarly, but not exposed to radiation.
High Fe: The high iron diet consisted of standard AIN93G
chow (45 mg iron/kg; Research Diets, New Brunswick, NJ)
supplemented with ferric citrate to 650 mg iron/kg.
Both: Animals received the high Fe diet and radiation
treatments.
200
0
Crewmembers on spaceflight missions are exposed to several
unusual environmental stressors, including microgravity and
chronic low dose radiation, as well as a diet of preserved
food. Dietary factors and exposure to radiation are aspects of
spaceflight that can be modeled in ground experiments. In this
experiment, we examine the effects of high dietary iron and
low dose gamma radiation, individually and combined, on the
gene expression of key metabolic enzymes.
METHODS
Drug metabolizing genes were differentially
affected by treatments
CONCLUSION
ACKNOWLEDGEMENTS
The authors thank Dr. Sara Zwart for animal tissues from her study (Evaluation of
the combined effects of gamma radiation and high dietary iron on oxidative damage
and antioxidant status in rats), Ms. Kami Faust and Mr. Andrew Hood for technical
assistance (Wyle Enterprises and NASA Career Exploration Program, respectively).
The JSC Human Health and Countermeasures Division Core Laboratories provided
some necessary instrumentation. Funding was provided to V. Wotring by NASA
JSC Human Research Program.