Immunohistochemical study of myoglobin and oxidative injury-related markers in the kidney of methamphetamine abusers

Legal Medicine 5 (2003) 42–48 www.elsevier.com/locate/legalmed

Immunohistochemical study of myoglobin and oxidative injuryrelated markers in the kidney of methamphetamine abusers Akiko Ishigami, Itsuo Tokunaga, Takako Gotohda, Shin-ichi Kubo* Department of Legal Medicine, School of Medicine, The University of Tokushima, 3-18-15 Kuramoto, Tokushima 770-8503, Japan Received 17 December 2002; received in revised form 10 January 2003; accepted 14 January 2003

Abstract It is known that methamphetamine (MA) causes rhabdomyolysis, myoglobinuria, and acute renal failure. We conducted an immunohistochemical study on the kidney of 22 forensic autopsy cases in which MA had been detected. Myoglobin was positive in 17 cases. The concentration of the blood MA in the myoglobin-positive cases (8.39 ^ 3.43 mmol/dl) was higher than -negative cases (0.198 ^ 0.076 mmol/dl). And, the 70 kDa heat shock protein (HSP70), 8-hydroxy-20 -deoxyguanosine (8-OHdG), 4-hydroxy-2-nonenal (4-HNE), and Cu/Zn superoxide dismutase (SOD) were also stained positively in five, ten, 11, nine cases of examined, respectively. In addition, 80% of HSP70-positive cases were myoglobin-positive. Myoglobin was also observed in 60% of 8-OH-dG-positive, in 82% of 4-HNE-positive, and in 78% of SOD-positive cases, respectively. Therefore, myoglobin rather than MA itself might induce oxidative damage. From these results, it was considered that MA abuse had caused the skeletal muscle damage before death. In forensic autopsy cases of drug abusers, the antemortem situation is not often known. The present research suggested that in addition to the measurement of the concentration of MA, immunohistochemical staining of myoglobin, HSP70, 8-OH-dG, 4-HNE, and SOD offers important information for the diagnosis of MA poisoning. q 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Methamphetamine; Kidney; Myoglobin; Oxidative injury; Immunohistochemistry

1. Introduction To diagnose the cause of a methamphetamine (MA) abuser’s death, the concentration of MA is the most important information. It was reported that a lethal concentration of MA in blood is 3.0 mmol/dl [1]. However, it is difficult to judge the cause of death when the concentration of MA is under the lethal level [2,3]. There have been many studies about the relation between cardiac lesions of MA abusers and the cause * Corresponding author. Tel./fax: þ 81-88-633-7084. E-mail address: [email protected] (S. Kubo).

of death [4,5]. On the other hand, it was reported that various drugs of abuse cause rhabdomyolysis [6 – 8]. MA also causes rhabdomyolysis [8 –10], myoglobinuria, and acute renal failure [11 –14]. Myoglobin is the heme protein in cardiac and skeletal muscle, and transports oxygen from the blood to the tissues. When the muscle is injured (cardiac infarction and trauma, etc.), it separates easily from the tissue, and comes out in blood and urine. Therefore, the measurement of myoglobin is used to diagnose muscle injury. Heat shock proteins (HSPs) are molecular chaperones that are induced by various stresses such as heat stress, inflammation, oxidative injury, and ischemia [15 – 19]. The 70 kDa HSP

1344-6223/03/$ - see front matter q 2003 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S1344-6223(03)00005-1

A. Ishigami et al. / Legal Medicine 5 (2003) 42–48

(HSP70) is especially effective to protect cells against heat stress. As one of the proto-oncogenes, c-fos plays a key role in the proliferation and differentiation, and is induced by various stresses [20 – 23]. Tumor necrosis factor (TNF) is a proinflammatory cytokine and is released from the kidney in response to ischemia and reperfusion [24,25]. 8-Hydroxy-20 deoxyguanosine (8-OH-dG) is a sensitive and useful marker of oxidative DNA damage, and is induced by ischemia-reperfusion-injury, and various carcinogens [26 – 30]. 4-Hydroxy-2-nonenal (4-HNE) is a major product of endogenous lipid peroxidation as a result of free radical attack [26,28,31,32]. In addition, Cu/Zn superoxide dismutase (SOD) is the most common scavenger and protects organisms against oxidative damage by free radicals [33 – 35]. In this study, we conducted an immunohistochemical study of the kidney in forensic autopsy cases in which MA had been detected, using the antibodies to myoglobin, markers for various stress and oxidative damage.

2. Materials and methods Twenty-two forensic autopsy cases with MA detected in the blood and/or the urine were examined as the MA-related cadavers. Based on the autopsy diagnosis, the cases were examined separately for two groups; intoxication and other causes of death. The total concentration of MA and amphetamine was expressed as the concentration of MA (mmol/dl) [1]. Table 1 shows the descriptions of the examined cases. Both kidneys were fixed in phosphate-buffered formalin, embedded in paraffin and sectioned at 5 mm. Hematoxylin-eosin (HE) was used as the conventional stain. Immunostaining was performed with antibodies against myoglobin (1:200, Dako, Denmark), the 70 kDa heat shock protein (HSP70, 1:1000, SigmaAldrich, USA), c-fos gene product (c-Fos, 1: 300, Oncogene Research Products, USA), tumor necrosis factor-a (TNF-a, 1:200, G-T Research Products, USA), 8-hydroxy-20 -deoxyguanosine (8-OH-dG, 1:250, QED Bioscience Inc., USA), 4-hydroxy-2nonenal (4-HNE, 1:400, Alpha Diagnostic Int., USA), and superoxide dismutase Cn/Zn enzyme (SOD, 1:200, OXIS Health Products Int., USA). The immunostaining was carried out using the AutoProbe

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IIe staining kit (Biomeda, USA) and MicroProbee system (FisherBiotech, USA) according to the manufacturer’s instructions based on the streptavidin-biotin complex technology with diaminobenzidine chromogen. The pathological findings of the kidneys were observed separately, at glomerulus, proximal tubule, and distal tubule. The concentration of MA in the blood or the urine was compared with immunopositive cases and immunonegative cases. Statistical analysis was performed with a nonparametric test, the Mann –Whitney U-test.

3. Results In conventional staining, the HE stain revealed the exfoliation of the renal tubule epithelium to the lumen, enhancement of eosinophilic staining in the renal tubule epithelium (in ten cases), and the appearance of the cast (in two cases). The results of the immunohistochemical study are shown in Table 2. The staining for c-Fos and TNF-a were negative in all cases. The staining for myoglobin was positive in 17 cases, and HSP70 was positive in five cases. The staining for 8-OH-dG was positive in ten cases, 4HNE was positive in 11 cases, and SOD was positive in nine cases. Myoglobin was positive-stained at tubular cells and cast (Fig. 1a). 8-OH-dG was positive-stained at the nuclei of tubular cells and glomeruli (Fig. 1b). The staining for HSP70, 4-HNE, and SOD were positive at tubular cells (Fig. 1c), and glomeruli in several cases. There was no clear relation between immunoreactivity for these antibodies and characteristics such as age, gender, postmortem duration, and the presence of alcohol drinking. We also tried to examine the relation between the presence of an abnormal behavior or hyperthermia and immunoreactivity. However, it was not possible to compare them because there were many cases with an uncertain antemortem situation. We examined the relation between immunoreactivity and the concentration of MA in the blood and the urine. The concentration of the blood MA in the myoglobin-positive cases (8.39 ^ 3.43 mmol/dl) was significantly higher than that of -negative cases (0.198 ^ 0.076 mmol/dl) (P ¼ 0:0011). However, no significant difference was found in other stains

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Table 1 The descriptions of examined cases (ND; not detected, NT; not tested, ?; unknown) Case No.

M F M M F M M M M M M M F F F M M M M M M M

Age (years)

22 18 28 43 19 23 30 29 35 30 26 47 39 23 60 35 47 41 51 34 39 54

Cause of death

Intoxication Intoxication Drowning Intoxication Intoxication Bleeding Hypothermic death Intoxication Intoxication Intoxication Intoxication Gunshot wound Burn Intoxication Drowning Intoxication intoxication Brain contusion Bleeding Intoxication Intoxication Intoxication

Postmortem duration

14 h 15 h 4 days 9h 1 day 10–14 h 21 h 20 h 32 h 8h 16 h 6h 17 h 16 h 29 h 24 h 12 h 12 h 12 h 8h 33 h 13 h

Concentration of MA (mmole/dl) Blood

Urine

0.503 11.4 ND 3.34 8.73 0.208 0.462 14.6 3.66 2.17 12.0 ND 2.47 5.87 0.210 1.58 4.03 þ (qualitative) 0.740 53.6 1.14 0.110

11.3 NT þ (qualitative) 76.0 58.8 0.318 21.6 78.8 119 NT 2.02 1.40 13.2 49.8 3.28 NT 367 1.30 12.9 693 7.76 11.1

Abnormal behavior

Hyperthemia

Alcohol

þ ? þ þ (convulsion) ? þ ? þ þ þ 2 2 ? 2 þ ? ? 2 þ ? þ

þ ? ? þ þ ? ? þ þ þ ? 2 ? þ ? þ ? ? þ þ ? þ

2 ? 2 2 ? 2 2 ? ? ? 2 2 2 2 2 2 2 þ 2 2 ? þ

A. Ishigami et al. / Legal Medicine 5 (2003) 42–48

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

Gender

A. Ishigami et al. / Legal Medicine 5 (2003) 42–48

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Table 2 The results of immunohistochemical staining (G; glomerulus, P; proximal tubule, D; distal tubule, þþ ; strongly positive, þ ; positive, 2; negative) Case No.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

Myoglobin

HSP70

8-OH-dG

4-HNE

SOD

G

P

D

G

P

D

G

P

D

G

P

D

G

P

D

2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2

2 2 2 þ þ þþ þþ þ 2 þ þþ þ þ þ þ þ 2 þ 2 þ þ 2

2 þ þ þ þþ þþ þþ þ 2 þ þþ 2 þþ þ þ þ 2 þ 2 þ 2 2

þ 2 2 2 2 2 2 2 2 2 þ 2 2 2 2 2 2 2 2 2 2 2

2 2 2 2 2 2 þ 2 2 þ þ 2 2 2 2 2 2 2 2 2 þ 2

2 2 2 2 2 2 þ 2 2 þ þ 2 2 2 2 2 2 2 2 2 2 2

2 2 2 þ þ 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2

2 2 þ þ þ þ 2 þþ þ 2 2 2 2 2 2 2 þ 2 þ 2 þ þ

2 2 2 þ 2 2 2 þ 2 2 2 2 2 2 2 2 2 2 2 2 2 2

2 2 2 2 2 2 þ 2 2 þ 2 2 2 2 2 2 2 2 þ 2 2 2

2 2 2 2 2 2 þ 2 2 þ þ þ þ þ þ 2 2 þ þ þ 2 2

þ 2 2 2 2 2 þ 2 2 þ þ þ þ 2 þ 2 2 2 þ þ 2 2

2 2 2 2 2 2 2 2 2 2 2 2 þ 2 2 2 2 2 2 2 2 2

þ þ 2 2 2 2 2 2 2 2 þþ þ 2 2 þþ þ 2 þ þ 2 2 2

2 þ 2 2 2 2 2 2 2 2 þ 2 2 2 2 2 2 2 2 2 2 2

between immunopositive cases and -negative cases (P . 0:05) (Table 3). The interrelation of immunoreactivity for each antibody was shown in Table 4. In five HSP70positive cases, four were myoglobin-positive (80%). In addition, in the 8-OH-dG (ten cases), 4-HNE (11 cases), and SOD-positive (nine cases), staining for myoglobin was positive in six cases (60%), nine cases (82%), and seven cases (78%), respectively.

4. Discussion To diagnose forensic autopsy cases as MA intoxication, the concentration of MA in blood and urine is very important [1]. On the other hand, the cause of one’s death is occasionally diagnosed as intoxication from the autopsy findings even if the concentration of MA does not reach the lethal dose [4, 5]. Recently, it was reported that the MA-abuser develops rhabdomyolysis, myoglobinuria, and acute

Table 3 The relation between each immunoreactivity and the concentration of methamphetamine in the blood Marker

Myoglobin Positive Negative HSP7O Positive Negative 8-OH-dG Positive Negative 4-HNE Positive Negative SOD Positive Negative

Concentration (mean ^ standard error mmol/dl) Blood

Urine

8.39 ^ 3.43 0.198 ^ 0.076

136 ^ 63.5 49.3 ^ 44.8

3.26 ^ 2.21 7.37 ^ 3.49

10.7 ^ 4.12 129 ^ 53.3

4.06 ^ 1.60 8.21 ^ 4.72

117 ^ 44.6 88.5 ^ 75.7

8.61 ^ 5.77 4.48 ^ 1.49

81.0 ^ 68.2 130 ^ 48.4

3.61 ^ 1.79 8.16 ^ 4.30

6.49 ^ 2.14 164 ^ 64.2

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Fig. 1. Positive immunohistochemical staining ( £ 100) a; myoglobin, b; 8-OH-dG, c; SOD (a, c; Case No. 11, b; Case No. 8).

renal failure [11 – 14]. Therefore, to investigate the meaning of the pathological findings of the kidney, we undertook an immunhistochemical study of the kidney in forensic autopsy cases in which MA had been detected, using the anti-myoglobin antibody. Fifteen of 20 cases (75%) in which MA was detected in blood were myoglobin-positive. Thirteen

of 18 cases (72%) in which MA was detected in urine were also myoglobin positive. Myoglobin was positive at a high frequency in MA-related cadavers. The concentration of the blood MA in the myoglobinpositive cases (8.39 ^ 3.43 mmol/dl) was significantly higher than that of -negative cases (0.198 ^ 0.076 mmol/dl) (P ¼ 0:0011). Therefore, it was considered that the appearance of myoglobin was related to the concentrations of MA. It was reported that MA causes rhabdomyolysis [8 – 10]. When we examined the immunoreactivity of myoglobin in five of 22 MA-related cases, myoglobin in the skeletal muscle was decreased in five of all cases (data not shown). Therefore, it is considered that there are some relations between myoglobin in the kidneys and the MA-induced muscular injury. The possibility of rhabdomyolysis or skeletal muscle damage was suspected. Immunostaining of HSP70, c-Fos, and TNF-a were performed. In this study, an immunostaining of HSP70 was positive in five cases (23%), but neither cFos nor TNF-a was immunostained. It was reported that psychostimulant drugs cause hyperthermia [36]. For two cases among five that were HSP70-positive, the presence of hyperthermia was confirmed antemortem (for the three remaining cases, it was not certain). This result suggests these cases might have had conditions to cause the expression of HSP70, i.e. heat stress [15,17]. Oxidative injury can occur when myoglobin invades the kidney [14,33,37– 39]. Therefore, 8-OHdG, which is an index of oxidative DNA damage [26 – 30], 4-HNE, which is a major product of endogenous lipid peroxidation [26,28,31,32], and SOD, which decomposes superoxide [33 – 35], were observed immunohistochemically. These oxidative injury-related markers were positive-stained in 40 – 50% of cases. This result might suggest that oxidative injuries (DNA damage, lipid peroxidation) were present. The relation between immunoreactivity and the concentration of MA in blood and urine was examined. However, in immunoreactivities of 8-OHdG, 4-HNE and SOD, there was no significant difference in the concentration between the immunopositive and -negative cases (P . 0:05) (Table 3). Therefore, these oxidative changes might relate to factors other than the concentration of MA. To reveal the factor that relate these oxidation,

A. Ishigami et al. / Legal Medicine 5 (2003) 42–48

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Table 4 The interrelation of immunoreactivity for each antibody Markers

Myoglobin HSP70 8-OH-dG 4-HNE SOD

Myoglobin

HSP70

8-OH-dG

4-HNE

SOD

n

(%)

n

(%)

n

(%)

n

(%)

n

(%)

– 4/17 6/17 9/17 6/17

4/5 – 1/5 3/5 1/5

(80)

(24) (36) (53) (36)

6/10 1/10 – 1/10 1/10

(60) (10)

9/11 4/11 1/11 – 6/11

(82) (36) (9)

7/9 2/9 1/9 7/9 –

(78) (22) (11) (78)

(20) (60) (20)

the immunohistochemical findings of MA-related cadavers were investigated in detail as follows. The number and ratio of each immunopositive cases was compared according to the cause of death: between the MA intoxication (14 cases) and other causes of death (eight cases). However, there was no obvious difference between the two. The interrelation of immunoreactivity for each antibody showed that myoglobin-positive cases included HSP70, 8-OH-dG, 4-HNE, and SODpositive cases in high ratio (Table 4). Recently, it was reported that oxidative injury was one of mechanisms which tissues and cells were damaged by MA [40,41]. On the other hand, it was also reported that severe muscular injury induced appearance of myoglobin and SOD-expression in the kidney [34]. From these results, it was considered that the positive staining of 8-OH-dG, 4-HNE, and SOD was greatly related to the presence of myoglobin rather than MA itself. In conclusion, it was considered that the MA induced the appearance of myoglobin from the skeletal muscle to the kidney, and myoglobin but not MA caused the oxidative damage. In forensic autopsy cases of drug abusers, the antemortem situation is not often known. The present research suggested that in addition to the measurement of the concentration of MA, immunohistochemical staining of myoglobin, HSP70, 8-OH-dG, 4-HNE, and SOD offers important information for the diagnosis of MA poisoning.

Acknowledgements The authors are deeply grateful to Prof. Noriyuki

(10) (10)

(55)

Tanaka of the University of Occupational and Environmental Health, and Prof. Yasuo Bunai of Gifu University School of Medicine for their generous supply of important samples.

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