Introduction

In 1985, a member of our group serendipi-tously achieved unexpected data on MPTP toxicity by demostrating, for the first time, that a compound, diethyldithiocarbamate (DDC), potentiates MPTP toxicity in the mouse model (Corsini, 1985). The DDC-induced enhancement of MPTP toxicity in mice has been extensively confirmed by numerous reports in which Authors give the results of their studies on MPTP metabolism in general and on MPP+ kinetics (the toxic metabolite) in particular, pursuing the definition of the enzymes responsible for the neurotoxic versus neuroprotec-tive pathways in the liver and brain as well.

More recently, the discovery of the occurrence of cytochrome P450 in the brain and in DA neurons as well, the function of which is still unknown (Warner et al., 1994), led to a new hypothesis in this respect (Corsini et al., 2002). In particular, cytochrome P450 2E1 (CYP 2E1) was identified in the rat brain and in DA neurons of the substantia nigra as well (Watts et al., 1998) and, at the same time, DDC was found to be a fairly specific inhibitor of this isozyme (Stott et al., 1997).

In order to understand the role played by CYP 2E1 in the DDC-induced enhancement of MPTP toxicity in mice, we studied the effects of diallyl sulfide (DAS) and pheny-lethylisothiocyanate (PIC), two specific inhibitors of CYP 2E1 enzymatic activity (Brady et al., 1991; Nissbrandt et al., 2001), on MPTP toxicity. CYP 2E1 knockout mice were challenged with MPTP or the DDC-combined treatment.

Materials and methods

Animals

Male C57/bl mice (Harlan, Italy), 8 weeks old and weighing 20 to 24 gr, were kept under environmentally controlled conditions (12hrs light/dark cycle with light on between 07.00 and 19.00 hrs; room temperature +21°C) with food and water ad libitum. The animals were treated in accordance with the Guidelines for Animal Care and Use of the National Institutes of Health. The experiments described in this article were formally approved by the Committee for Scientific Ethics of the University of Pisa.

Knockout mice

Male Cyp 2e1 knockout mice (129/SV-Cyp 2e1tm1Gonz) (Cyp 2e1—/— Stock number: 002910) and their wild type counterparts (129S1/SvlmJ) (Cyp 2e1+/+ Stock number: 002448) were obtained from The Jackson Laboratory (Bar Harbor ME, USA). Cyp2e1 (—/—) mice in 129/Sv-Ter background were generated in the Gonzalez laboratory (Lee et al., 1996) back-crossed four times into the wild type 129/Sv-Ter strain. These animals were screened for viral infection by Charles River Laboratories and all tests were negative. Confirmation of the Cyp 2e1—/— status was confirmed by the absence of CYP 2E1 as determined by liver DNA PCR phenotyping by Charles River Laboratories.

Experimental protocol

Twelve mice per group were treated i.p. with either MPTP hydrochloride (36mg/kg) or distilled water.

The animals were pretreated i.p. with DDC (400 mg/kg) or DAS (25 mg/kg) or PIC (25 mg/kg) or the vehicle, one hr before MPTP administration. DDC was easily dissolved in distilled water, whereas the liquid DAS was given already dissolved in a mixture of DAS plus tween 80 plus propylene glycol plus distilled water in 1, 1.5 and 10 volume proportions, respectively. The liquid PIC plus distilled water and one drop of tween 80 was made up to the appropriate volume and then homogenized with an ultrasonic disrupter prior to the injection. DA has been measured according to Vaglini et al. (2004). For statistical evaluation ANOVA with Sheffe-F analysis was used.

Results

Striatal modification of DA in C57-bl

The effect of the acute administration of CYP 2E1 inhibitors on the dopamine (DA) content in the mouse striatum one hour before a single exposure to MPTP is shown in Fig. 1.

In agreement with previous data (Corsini et al., 1985), 7 days after the combined treatment (DDC + MPTP), striatal DA content fell to 8.9% of controls (10.3 ± 2.8 and 115.2 ± 4.5ng/mg protein, respectively). The animals treated with DDC alone did not show any change compared with control values. Figure 1 also shows the effects of pretreat-ment with DAS. The combined treatment

Controls Inhibitors MPTP DDC+MPTP DAS+MPTP PIC+MPTP

Fig. 1. Effect of DDC (400mg/Kg), DAS (25 mg/kg) and PIC (25 mg/kg) on striatal tissue levels of dopa-mine (DA) in MPTP-treated mice. The results are the mean ± s.e. of 3 experiments (n = 6-8 mice for each experiment). C57-bl were treated with MPTP (30 mg/kg i.p.) or saline 60min. after DDC, DAS or PIC. The animals were sacrified 7 days later. Respective controls received saline instead of inhibitors or MPTP. *p < 0.05 in comparison with control mice; **p < 0.05 in comparison with the animals treated with MPTP

Controls Inhibitors MPTP DDC+MPTP DAS+MPTP PIC+MPTP

Fig. 1. Effect of DDC (400mg/Kg), DAS (25 mg/kg) and PIC (25 mg/kg) on striatal tissue levels of dopa-mine (DA) in MPTP-treated mice. The results are the mean ± s.e. of 3 experiments (n = 6-8 mice for each experiment). C57-bl were treated with MPTP (30 mg/kg i.p.) or saline 60min. after DDC, DAS or PIC. The animals were sacrified 7 days later. Respective controls received saline instead of inhibitors or MPTP. *p < 0.05 in comparison with control mice; **p < 0.05 in comparison with the animals treated with MPTP

Table 1. Effect of DDC on striatal tissue levels of dopamine (DA) in wild type (SVI) and knockout (GONZ) (Cyp 2e1—/—) male mice treated with MPTP. The results are the mean ± s.e. of N(10-20) animals for each group. Wild type and knockout were treated with MPTP (30mg/kg i.p.) or saline solution 60min after DDC at the dose of

400mg/Kg i.p. The animals were sacrified 7 days later

Table 1. Effect of DDC on striatal tissue levels of dopamine (DA) in wild type (SVI) and knockout (GONZ) (Cyp 2e1—/—) male mice treated with MPTP. The results are the mean ± s.e. of N(10-20) animals for each group. Wild type and knockout were treated with MPTP (30mg/kg i.p.) or saline solution 60min after DDC at the dose of

400mg/Kg i.p. The animals were sacrified 7 days later

Treatments

(CYP 2E1+/+) DA (ng/mg prot.)

(CYP 2E1—/—) DA (ng/mg prot.)

%

Controls

114.1 ± 8.3

109.4 ± 9.8

DDC

105.8 ± 9.0

108.8 ± 4.1

MPTP

80.2 ± 4.1*

29

76.5 ± 6.3*

30

DDC + MPTP

48.7 ± 5.1**

57

73.7 ± 5.7***

32

*p<0.05 in comparison with control mice; **p<0.05 in comparison with MPTP-treated animals; ***n.s. in comparison with MPTP-treated animals

*p<0.05 in comparison with control mice; **p<0.05 in comparison with MPTP-treated animals; ***n.s. in comparison with MPTP-treated animals

(DAS + MPTP) reduced the striatal DA content to 7.2% whereas MPTP alone caused a reduction of only 45.0% compared with controls. (Control values were 110.2 ± 8.2ng/mg protein; 7.9 ± 1.0, 49.5 ± 4.4ng/mg protein DAS + MPTP and MPTP respectively). As shown in Fig. 1, PIC also potentiated MPTP toxicity. The combined treatment significantly reduced the striatal DA content in comparison with MPTP alone (p<0.05).

MPTP toxicity in CYP 2E1 knockout mice (GONZ)

The effect of MPTP treatment on the striatal DA content was studied in CYP 2E1—/— (GONZ) and wild type (SVI) male mice. As shown in Table 1, the single usual dose of 30 mg/kg i.p. produced a significant reduction in GONZ (30%) and SVI (29%) as well animals. The DDC pretreatment, at the usual time and dose schedule, completely failed to potentiate the DA fall in knockout mice (32%) whereas in wild type animals the combined treatment significantly enhanced the DA fall up to 57%.

Was this article helpful?

0 0
All About Alzheimers

All About Alzheimers

The comprehensive new ebook All About Alzheimers puts everything into perspective. Youll gain insight and awareness into the disease. Learn how to maintain the patients emotional health. Discover tactics you can use to deal with constant life changes. Find out how counselors can help, and when they should intervene. Learn safety precautions that can protect you, your family and your loved one. All About Alzheimers will truly empower you.

Get My Free Ebook


Post a comment