Nuclear Instruments and Methods in Physics Research B22 (1987) 217-222

North-Holland. Amsterdam

217

PIXE ANALYSIS OF TRACE METALS IN SELENIUM AND COPPER DEFICIENT MICE EXPOSED TO INFLUENZA VIRUS AND SALICYLATE

J.M. ANDRES, R.W. HURD, H.A. VAN RINSVELT and P.A. SMALL

Departments of Pediatrics (Gastroenterology), Food Science and Human Nutrition, Physics, and Medical Microbiology, University

of Florida, Gainesville, Florida 32611. USA

W. MAENHAUT and J. VANDENHAUTE

Institute for Nuclear Sciences, University of Gent, Belgium

Reye’s syndrome is an acute illness in children manifested by encephalopathy, fatty infiltration of liver, and thymic hypoplasia.

The syndrome usually occurs in a susceptible individual with a viral illness who has ingested salicylate. We previously investigated

the metal status of children with this syndrome; serum Se and Cu levels were noted to be decreased. Chronic aspirin treatment of

rats also produced alterations of serum Se, and liver Se and Cu. We now report our observations for an experimental model of

Reye’s syndrome. Analysis by PIXE of various metals in Se- and Cu-deficient mice exposed to virus and salicylate are discussed.

1. Introduction

Reye’s syndrome (RS), an acute illness in children, is manifested by encephalopathy; fatty degeneration of the liver, heart, and kidneys; and thymic hypoplasia [I - 31. The syndrome may be secondary to mitochon- drial injury following a toxic insult in a susceptible child with a viral illness. We previously reported that the possible pathogenetic mechanism contributing to the mitochondrial disfunction was alteration of the intracellular metal environment [4]. Decreased serum and hepatic selenium (Se) and copper (Cu) were demonstrated via particle-induced X-ray emmission (PIXE) in patients with RS, in addition to an increase in serum iron (Fe) and zinc (Zn) [4]. In a subsequent study using a rat animal model of RS, the hepatotox- ins 4-pentenoic acid and valproic acid produced similar disturbances in serum and liver concentrations of Se, Cu, Fe and Zn [.5]. Because of the possible association of RS with another potential hepatotoxin, acetyl- salicylic acid (Aspirin) [6], we then investigated metal balance using PIXE analysis in young rats chronically exposed to this commonly used metal chelator 171. The animals had decreased serum levels of Se, Zn and Fe compared to controls. Selenium was also decreased in liver as was Cu, and there was a trend toward in- creased hepatic calcium (Ca). In the present study, PIXE was used to further investigate the effect of Se- and Cu-deficient diets on several tissues in mice; only the results for liver and thymus will be reported at this time. Subgroups of mice received influenza A virus and sodium salicylate; histologic sections of liver were

0168-583X/87/$03.50 0 Elsevier Science Publishers B.V. (North-Holland Physics Publishing Division)

studied to assess the effect of these agents in animals on specific metal-deficient diets.

2. Experimental

2.1. Design and specimen selection

Young CD-1 male mice (n = lOS), 20 g initial weight (Charles River, MA), were divided into four main experimental Cu and Se groups (table 1). Animals fed deficient diets were also resupplemented with Cu sul- phate (5 mg/l) and sodium (Na) selenite (0.1 mg/l) added to demineralized, distilled water. All animals were housed in stainless steel metabolic cages and allowed ad libitum access to water and special diets (ICN Nutritional Biochemicals, Cleveland, Ohio). Control mice were fed Rodent Lab Chow (#5001, Ralston Purina Co., St. Louis, MO). After 6 weeks, mice were inoculated with influenza A virus (H,N,) via the intranasal route [S]. Intraperitoneal Na salicy- late (#53007, Sigma Chemical Co., St. Louis, MO) was administered daily (50 mg/kg) to the appropriate groups of mice (table 1) starting 6 days after exposure to virus, and continued for one week prior to sacrifice. Liver, thymus, spleen, heart and kidney were removed using stainless steel dissection tools. All organs were rinsed in physiologic saline solution, blotted dry, weighed and lyophilized. A portion of liver from each animal was added to a formalin fixative and submitted for histologic evaluation. We report here the PIXE analysis of metal concentrations in liver and thymus, and Se levels in spleen.

II. BIOLOGICAL/MEDICAL APPLICATIONS

218 J.M. Andres et ul. I Truce metals in Se and Cu deficient mice

Table 1 Experimental design for Cu and Se mice (n = 8 for each subgroup) with subgroup exposure to influenza virus and salicylate~“.

Animal group Subgroups

(-1 Virus (+i Virus ( +) Virus (+ ) Salicvlate

Cu-deficient (+) CuSo,

Cu-deficient Se-deficient

Se-deficient (+) Na selenite

-cu + cu -v -cu+cu+v -Cu+Cu+VA -cu -v -cu+v -Cu + VA -Se-V -Se+V -Se + VA

-Se+Se-V -Se + Se + V -Se+Se+VA

“IV= influenza A virus; A = Na salicylate; (+) = exposure to virus/and salicylate

2.2. PIXE procedure

For the PIXE analysis, an internal standard was added, 3 targets were prepared and each target was bombarded with 40 PC, 2.4 MeV protons. Two differ- ent sample/target preparation procedures were ap-

plied. The “brittle fracture technique” was used for doping, homogenizing and target preparation of all liver samples. The thymus and spleen samples, on the other hand, were smaller (<lOmg) and the “wet digestion technique” was used. The PIXE analytical procedures were completed using the compact iso- chronous cyclotron at the University of Gent and have been previously described in detail [9-111.

3. Results and discussion

Particle induced X-ray emission (PIXE) analyses of trace metals in liver and thymus tissues are shown in figs. 1 and 2. These results are given as the percent of control for all groups of mice on different Se and Cu diets; whereas, numeric results and standard devia- tions are reported for control animals only (table 2). Statistical significance (p < 0.05) is noted for trace metals if three subgroups of a treatment group (e.g., -Q-V, -Cu +V, -Cu+ VA of group -Cu) are significantly different from controls. For the Cu-defici- ent mice, there was a significantly elevated liver (fig. 1) Fe concentration, but decreased hepatic Se, Br, and Rb. The highly significant increase in Fe was observed despite the finding of low Fe in the Cu- deficient diet (table 3). Hepatic accumulation of Fe in Cu-deficiency states has been previously reported [ 121. This observation may be related to inhibition of hemoglobin synthesis due to low Cu concentrations required for stimulation of the production of heme. Animals with Cu-sufficiency also demonstrated sig- nificantly low levels of tissue Se, Br, and Rb.

The Se-deficient and Se-resupplimented animals both had decreased liver Se, Br, Rb, and Mn, with the additional finding of low hepatic Zn noted in the Se-sufficient group. An increase in serum Zn and Fe

cl = -cu +cu

q =-Se+%?

l =pcoo5

t

T Br Rb Mfl

Fig. 1. Trace metal concentrations (pgig dry weight) as

percent of control in liver of four dietary treatment groups.

-Cu and -Se, deficient diets; -Cu + Cu and -Se + Se.

resupplemented diets.

levels [ 131, or a decrease in serum Se [14] and Cu [ 151, has been associated with promoting microbial infec- tions in addition to some viral diseases. Certain drugs such as salicylic acid modify the normal rise in serum Cu observed with influenza infection 1161. Salicylate treatment also results in a modification of prostaglan- din metabolism in Se-deficient animals [17]. Regula- tion of prostaglandin synthesis may be related to the subcellular distribution of glutathione peroxidase and

Se [18]. The thymus tissue (fig. 2) results were most signific-

ant for the markedly elevated Ca and Mn in Cu- deficient mice. For this same group there was also a

J. M. Andres et al. I Trace metals in Se and Cu deficient mice 2lY

IOO-

50-

w El = -cu

q = -Se

q = -Se+%

* = PC005

r-l I7

0

I CU Zn w *

Fig. 2. Trace metal concentrations (pglg dry weight) as

percent of control in thymus of four dietary treatment

groups.

low level of Rb and Cu. Significantly decreased Rb Despite the statistically significant changes for levels was seen in Cu-sufficient animals. Both Se treatment of hepatic and thymic Se, no group (e.g., -Cu + Cu) groups had decreased Rb in the thymus; elevated Zn or subgroup (e.g., -Cu + Cu + V) variation in Se was levels were noted in Se-sufficient mice, and there was discovered (fig. 3). Not shown in fig. 3 is the higher Se the expected decrease in Se for the Se-deficient ani- concentration for all five organs examined (liver, thymus, mals. The high thymic Ca level in the Cu-deficient spleen, heart. kidney) in the four Se treatment groups: 19 mice was observed despite the low Ca in the diet, of 20 tissues had more Se when exposed to virus

Table 2

Concentration (p&/g dry weight, m k SD) of elements in

liver and thymus from controls.

Element

K

Ca

Fe

cu

Zn

Se

Br

Rb

Mn

Liver

10 390 k 340

1102 19

860 t 200

14.2+ 0.6

89.45 4.6

4.6-c 0.3

4.72 0.4

30.0+ 2.6

4.2t 0.4

Thymus

13 200 _f 2300

1165 21 235 2 50

4.1 + 1.2

58.4 5 8.4

0.6 t 0.2

25.12 4.2

0.7 !I 0.2

although this element was available well above the recommended dietary allowances (RDA). The mech- anism for maintenance of high Ca levels in the thymus is not understood. However, it is known that calcium levels are under strong homeostatic control. Thymic involution does occur in patients with Reye’s syn- drome [3]; this may be related to the level of specific trace metals and perhaps their effect on cellular im- munity. Both Zn deficiency and cadmium administra- tion have been noted to cause atrophy of the thymus [19]. The significance of elevated Mn in the thymus of animals for both Cu treatment groups is unknown. Hepatic Mn levels are low in children with Reye’s syndrome [20] and in animals treated with pentenoic acid [5], a chemical causing a Reye-like disease [21].

Table 3

Concentration (PgIg dry weight) of elements in laboratory chow, Cu-

deficient. and Se-deficient diets. Comparison with mice and rat RDAs”‘.

Element Lab chow -cu -Se RDA

Mice Rat

K 14 600 6040

Ca 13 000 5960

Fe 269 48

cu 14 1

Zn 81 30

Se 0.41 <o

Br 9.6 1

Rb 8.61 0

Mn 56 68

.68

.12

.Y4

.24

12 300 2000 3600

13 500 4000 5000

355 25 35

11 4.50 5.00 36 30 12

< 0.12 O.lU

1.52

2.51

11 45 SO

“‘-Cu = Cu-deficient, -Se = Se-deficient, RDA = recommended dietary al-

lowances.

II. BIOLOGICAL/MEDICAL APPLICATIONS

220 J.M. Andres et 01. I Truce metals in Se and Cu deficient mice

5

4

@I 3

ppm

dry

2

CmeanrfrSD)

Liver 1

- = Liver .“.,(( = Sp&..

---_-- = Thymus

V = Virus A = Salicylate

Spleen :I

c I-Cu+CuJ I -cu 1 I-Se+Sel 1 -Se 1

V VA V VA V VA V VA

Fig. 3. Mean selenium concentrations (ppm dry weight *SD) for liver, spleen, and thymus in all subgroups of four dietary

treatment groups. ([Se] = selenium concentration.)

Fig. 4. Fat accumulation, hepatocellular disruption, and dark inclusion bodies in this liver specimen from a selenium animal.exposed to influenza virus (H&E, x 400)

deficient

J.M. Andres et al. I Trace metals in Se and Cu deficient mice 221

Fig. 5. Liver of selenium-resupplemented animal exposed to influenza virus showing only cellular fat deposition (H&E. X 400).

compared to their respective controls. This is probably highly significant but subgroup statistical analyses have not as yet been completed. Our data would suggest a preferential retention of Se in tissues of virus-exposed animals. Selenium is known to modify the ability of influenza virus to replicate [14]. Furthermore, it is well established that Se depletion in the serum causes a decrease in glutathione peroxidase activity [22] which may lead to abnornal mitochondrial Ca release [23] and subsequent lipid peroxide damage of cellular membranes. Cu-deficiency may also contribute to re- duced glutathione peroxidase activity [24].

The Se- and Cu-deficient animals were markedly less active than controls. More clincial illness was noted in the animals fed the Cu-deficient diet; they exhibited heart enlargement, thymic hypoplasia, pal- lor, and sparse, coarse hair. The Se-deficient animals also had a small thymus but were generally more well-appearing. Interestingly, the livers of Se-deficient mice contained more fat; after exposure to influenza virus, the histology was markedly worse with fatty infiltration, hepatocellular disruption, and the pres- ence of dark inclusion bodies (fig. 4). These inclusions could represent calcified material, since Ca levels were extremely elevated in some animals with fatty livers. The livers of Se-resupplemented mice showed only fat deposition despite exposure to virus (fig. 5). This putative protective effect of Se may be beneficial to children who develop Reye’s syndrome.

Trace metal analysis of all mouse diets and RDA

(expressed as required dietary concentrations) are given in table 3. Since rats are the most frequently studied rodents, their dietary allowances for various trace metals are also included. Significant differences between the diets were observed; for some of the trace elements the concentrations in the Se- and Cu-defici- ent diets were below RDA values. The Cu-deficient diet also contained low concentrations of Se, in addi- tion to Ca, Fe, Zn, Rb, and Br. In contrast, the Se diet was deficient in Mn, Zn, Rb, and Br, along with the low Se. It should be noted that Ca and K were well above the RDA for mice even for the Cu- deficient diet. Therefore, this study also demonstrates that in liver and thymus the concentration of Se, Cu, Br, Rb, and Mn depends on the composition of the diet ingested by the experimental animal. It is obvious from our data that there is a large difference between the composition of synthetic diets and normal labora- tory chow (table. 3). Reliable conclusions can only be obtained concerning trace element interactions if the composition of control and deficient diets are similar for all other nutrients.

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