This study investigates how manganese (Mn) stimulates oxidative stress in manganism, a movement disorder caused by high Mn levels resembling Parkinson's disease. The researchers discovered that Mn speeds up the redox cycling of dopathiazines, dopamine derivatives present in the basal ganglia brain region. This redox cycling produces reactive oxygen species and leads to oxidative stress. Mn uniquely stimulated redox cycling of dopathiazines compared to other metals. The effect requires inorganic phosphate and suggests Mn forms a complex with two dopathiazine molecules and phosphate. This Mn-stimulated redox cycling may explain the neurological selectivity of manganism symptoms.

2. Research Project Title
Dopathiazines magnify Mn-
dependent oxidative stress
in Manganism

3. Authors
Tareq Hanna, Praneet Marwah and David Njus*
Department of Biological Sciences
Wayne State University

4. Abstract
Manganism is a condition caused by chronic high levels of Manganese. Interestingly,
this selectively disrupts signaling in the basal ganglia, so Manganism is a movement
disorder resembling Parkinson's Disease. The basal ganglia are a dopamine-rich region
of the brain, and we have discovered that MnCl2 speeds up the redox cycling of
dopamine-derived benzothiazines, called dopathiazines. This redox cycling reduces O2
to superoxide and hydrogen peroxide, so it leads to oxidative stress. This effect is only
seen with Mn and is not reproduced by other metal ions such as Fe, Cu, Zn, Co, Ca or
Mg. The Mn effect requires that inorganic phosphate be present, suggesting that the
phosphate is involved in stabilizing a Mn:dopathiazine complex. It is proposed that
these or similar endogenous dopamine derivatives may magnify Mn-dependent
oxidative stress accounting for the neurological selectivity of manganism.

5. Introduction
Catechols are known to form specific complexes with Mn, and these have been studied
as models for the water-splitting center in photosynthesis (Larsen et al., 1986). Like
dopamine and its derivatives, catechols can bind Mn and lead to redox reactions which are
known to lead to oxidative stress. We examined the effect of manganese on the redox
cycling activity of a numerous dopamine derivatives and catechol analogs to test this
possibility (Figure 1). In addition to suggesting a possible route for these pathological
effects, redox cycling allows us to screen for compounds that react readily with O2 and
interact with manganese.
The dopamine quinone reacts rapidly with thiols like cysteine so when dopamine is
oxidized it yields cysteinyl-dopamine. Both dopamine and cysteinyl-dopamine have
relatively high reduction potentials and oxidize slowly in the presence of O2. Our lab
discovered that reaction of cysteinyl-dopamine and other catechol/thiol adducts with
hypochlorite forms benzothiazine derivatives with redox cycling activities that are greatly
accelerated by Mn. These products, which we call dopathiazines, include DTM2, formed
from dopamine and cysteine, DTM1 (which is formed from dopamine and cysteamine) and
DTM0 (formed by 4-methylcatechol and cysteamine). These dopathiazines have a
characteristic of being sensitive to Mn-stimulation of two-equivalent redox cycling that
separates them from other dopathiazines.

6. Methodology/Experimental
Synthesis of Dopathiazines
Dopathiazines were synthesized by oxidizing a catechol (dopamine) in the
presence of a thiol (cysteine) using ceric ammonium nitrate. The product was
treated with NaOCl in HCl and then purified by chromatography through Dowex
50Wx8 and a C18 reverse phase column (SepPak).
Oxidation

7. Methodology/Experimental
Redox Cycling
Redox cycling occurs when a compound (Q) is repeatedly reduced and the reduced form
is reoxidized by O2. Redox cycling is observed as a decrease in the amount of O2 after the
addition of a reducing agent such as dithiothreitol or ascorbic acid. Redox cycling was
measured as the initial rate of O2 consumption following addition of either 2.5 mM
ascorbic acid or 0.5 mM dithiothreitol. Oxygen concentration was monitored in 4 ml of
buffer solution containing redox cycler and other additions as indicated at 37°C using a
Clark-type oxygen electrode.

8. Figure 1
Figure 1. Manganese stimulates dithiothreitol-driven redox cycling (a) but not
ascorbate-driven redox cycling (b). O2 concentration was recorded at 37°C in 4 ml of
0.2 M potassium phosphate, 1 µM EDTA, pH 7.4 containing DTM2 and MnCl2 as
indicated. Redox cycling was initiated by adding 0.5 mM dithiothreitol or 2.5 mM
ascorbic acid at the arrow.
0
50
100
150
200
250
300
-2 0 2 4 6
[O₂]
(µM) Time (minutes)
12.5 µM Mn
200 nM DTM2
Mn + DTM2
Ascorbate
0
50
100
150
200
250
300
-2 0 2 4 6
[O₂]
(µM)
Time (minutes)
5 µM Mn
800 nM DTM2
Mn + DTM2
Dithiothreitol

9. Figure 2
0
50
100
150
200
No
metal
Mn Fe Cu Co Zn Mg Ca
Rate
(µM
[O₂]/min)
Figure 2. Manganese uniquely stimulates dithiothreitol-driven redox cycling. Redox
cycling by 200 nM DTM2 following addition of 0.5 mM dithiothreitol was recorded as
in Figure 1 in the presence of 10 µM concentrations of MnCl2, FeCl3, CuCl2, CoCl2,
ZnSO4, MgCl2, CaCl2 or no metal (1 µM EDTA). Rates of redox cycling were
determined from the initial slope following dithiothreitol addition. Each bar shows
the average (±SD) of three replicate samples.

10. Figure 3
0
20
40
60
80
100
120
140
160
DTT no
Pi Mn
DTT Pi
Mn
DTT no
Pi
DTT Pi Asc no
Pi
Asc Pi
Rate
(µM/min)
Figure 3. Inorganic phosphate is required for Mn-stimulated redox cycling but not for
ascorbate-driven redox cycling or for dithiothreitol-driven redox cycling in the absence of
Mn. The rate of redox cycling of 1 µM DTM2 was measured in 2 ml of 50 mM Hepes(KOH),
pH 7.4 and 2 ml of either 0.2 M potassium phosphate, 1 µM EDTA, pH 7.4 (Control) or H2O
(no Pi) in the presence or absence of 10 µM MnCl2. Redox cycling was initiated by adding 2.5
mM ascorbic acid or 0.5 mM dithiothreitol. Each bar shows the average (±SD) of three
replicate samples.

11. Figure 4
0
20
40
60
80
0 5 10 15
Rate
(µM
[O₂]/min)
[Mn] (µM)
665 nM DTM2
333 nM DTM2
166 nM DTM2
0 DTM2
a)
0
50
100
0 200 400 600 800
Rate
(µM
[O₂]/min)
[DTM2] (nM)
12.5 uM Mn
5 uM Mn
2.5 uM Mn
1.25 uM Mn
0.5 uM Mn
0 Mn
b)
Figure 4. Dependence of Mn-stimulated redox cycling on concentrations of
Mn and redox cycler. a and b) Rates of redox cycling were measured from
traces as shown in Figure 1a with DTM2 and MnCl2 at the indicated
concentrations. Lines show rates predicted by a kinetic model assuming
formation of a 2:1 complex of DTM2 to MnCl2.

12. Redox Cycling Mechanism
A) Hypothesized
mechanisms of redox
cycling driven by
dithiothreitol and
ascorbic acid. One-
equivalent cycling driven
by ascorbic acid avoids
the slow oxidation of the
fully reduced compound,
which requires either
superoxide or
comproportionation.
B) Mn stimulates two-
equivalent redox cycling
by facilitating oxidation of
the fully reduced
compound in a 2:1
complex.

13. Conclusions
1. Mn stimulates dithiothreitol-driven redox cycling of dopathiazines.
2. The effect on dithiothreitol-driven redox cycling is unique to manganese
and is not reproduced by Fe, Cu, Ca, Zn, Mg, or Co.
3. Inorganic phosphate is required for the Mn effect.
4. The effect of Mn on redox cycling of dopathiazines suggests the formation
of a complex consisting of 1 Mn, 2 dopathiazines and inorganic phosphate
5. The stimulation by Mn of redox cycling of dopathiazines or similar
dopamine derivatives may account for the unique vulnerability of the
dopamine-rich basal ganglia to Mn toxicity.

14. Acknowledgments
We thank Gijong Paik, Christopher Issa, Christopher Jemison, Muhammad
Qureshi, and Dr. Eduardo Palomino for assistance with this work.