Biochem. SOC. Symp. 6 I, 153- I 6 I
Printed in Great Britain
Biological and biochemical effects
of air pollutants: synergistic effects
of sulphite
Susanne Hippeli and Erich F. Elstner
Lehrstuhl fur Phytopathologie, Labor fur angewandte Biochemie, Technische
Universitat Munchen, 853 50 Weihens tephan, Germany
Abstract
Air pollution has become a major public and political concern since the
beginning of industrialization, particularly motor exhaust over the past three
decades. Epidemiological studies, together with clinical trials and experiments in
exposition chambers (including biochemical model reactions), have contributed to
our knowledge of potential dangers and increased our understanding of the
corresponding mechanisms and dose-response effects. Comparison of the threatening reports that appear almost daily in the press with the digest of over 800
scientific publications allows the statement that the impact of ozone and nitric
oxide on the health and performance of plants and animals is widely overestimated
and appears to be used as a political instrument. In contrast, the combination of
SO, with soot or asbestos particles may represent an underestimated toxic
potential.
In this communication, we shall concentrate on basic redox mechanisms
involving SO2 and important target molecules, as well as looking at the cooperative effects of sulphite and soot particles.
Introduction
Air pollution research has tremendously increased in the past two decades,
accompanied by the publication of a wealth of data. To mention just a few titles:
‘Responses of Plants to Air Pollutants’ [ 11, ‘Introduction to Environmental
Toxicology’ [ 2 ] , ‘Air Pollution and Forests’ [3], ‘Effects of Gaseous Air Pollution
in Agriculture and Horticulture’ [4], ‘Gaseous Air Pollutants and Plant Metabolism’ [5], ‘Air Pollution by Photochemical Oxidants’ [6] and ‘Air Pollution and
Plant Metabolism’ [7]. Several reviews emphasizing the importance of oxygen
radicals in air pollution have also appeared recently [8-lo].
I53
I54
Effects of air pollutants
The role of oxidative processes in air pollution has already been reported in
the last century when the production of acid rain was recognized as an oxidation
by atmospheric hydrogen peroxide of sulphur dioxide stemming from coal
combustion [1I].
SO, emissions are of special interest as the main component of the acid
(London-type) smog and as a monocausal trigger of forest decline ('Waldsterben')
in SO2-polluted areas.
Certain trace gases stemming from industry, traffic and agriculture are a
growing threat, especially for the industrialized countries all over the world. In the
last few years especially, automobile exhaust has advanced to become a focus of
public and political discussions and decisions. Airborne soot particles stemming
both from industrial incinerators and diesel engines are discussed as potential
carcinogens, allergens and/or inducers of respiratory diseases. The combinations of
SO, and soot from coal burning have been epidemiologically shown to be responsible for thousands of deaths during several severe smog episodes both in London
and in New York not too long ago. Several reports have appeared concentrating
on biochemical mechanisms underlying these synergisms [10,12-141.
Significant health-threatening effects brought about by gases or air-borne
particles may in the first line be seen in the cooperation of catalytically active
particles with SO,. This will be outlined in more detail below.
Reactions of SO2 with biomolecules
SO, is produced during combustion of organic materials, especially coal. SO,
is a highly water-soluble gas which is rapidly hydrated forming sulphite (SO3'-)
[15]. Principally, SO, or HSO; preferentially reacts with the following types of
molecules: (a) aldehydes and ketones, where hydroxysulphonates are formed,
which in turn are inhibitors of several enzymes; (b) olefines, where sulphonic acids
are formed and the double bond is lost; (c) pyrimidines under formation of
dihydrosulphonates, which may have mutagenic effects; (d) disulphides during
formation of S-sulphonates, where the S-S bridge is split; and (e) superoxide
(02'-)
under formation of the very reactive bisulphite radical:
O2'-+ S032-+2H+
so3'-+
H202
In this reaction, superoxide may be substituted by transition metal ions:
SO3'- is a powerful initiator of several radical chain processes such as lipid
peroxidation (see below). Sulphite modifies the cellular energy metabolism in
mammalian tissues by decreasing the cellular ATP pool; this may be due to
changing the function of pyridine and flavin nucleotides [16]. Sulphite oxidase, an
enzyme absent in human lung tissue, oxidizes sulphite. Thus, if sulphite is not
metabolized, it may be oxidized via radical chain mechanisms.
Plant damage by SO2
Plant damage, caused by SO,, is oxygen and light dependent. Typical SO,
effects are the rapid inhibition of photosynthesis before direct symptoms such as
I55
S. Hippeli and E.F. Elstner
bleaching of chlorophyll can be observed. The bleaching of chlorophyll is the most
prominent effect of SO,. SO2,or its aqueous solution sulphite, can be oxidized in
[15]. Limited N A D + availability in the stroma
the presence of superoxide (02'-)
of chloroplasts results in the formation of superoxide via 'over'-reduction of the
electron transport system and subsequent electron channelling to oxygen. Free
radicals generated during sulphite autoxidation attack cellular membranes via lipid
(L) peroxidation and subsequent chlorophyll (Chl) bleaching:
so;- +
02.-
+2Hf
SO,'-+ LH
+ SO3'-
+ HSO,
+ H202
+ L'
+ 0, + LOO'
LOO' + L H + L O O H + L'
L'
Me
LOOH
LO'
+ LO'+
OH-
+ Chl,,d+LOH + Chl,,
The bleaching of chlorophyll by lipid peroxidation is only observed if SO3,- is
simultaneously present. Neither L O O H nor
alone can destroy the green
colour of chlorophyll.
The generated H 2 0 2 is thought to be responsible for the inactivation of
ascorbate peroxidase, glutathione reductase and enzymes of the Calvin cycle, such
as phosphatases [17,18]. Uncoupling of oxidative phosphorylation results in
insufficient reoxidation of NADPH, and thus causes inhibition of photosynthesis
in the light due to a lack of ATP.
Cooperative effects of sulphite and soot particles
The discussion on the potential toxicity of diesel exhaust is of growing
importance, especially due to the increasing truck traffic in Middle Europe since
the opening of the East. Much of the threat of Otto-engine exhaust has been
taken away by the introduction of the three-way catalytic converter. Such a redox
converter is not applicable for diesel engines due to the different incineration
concept: only oxidative reactions are relevant for the diesel catalyst. Of special
importance, however, are the particulate emissions which are 30-70 times higher
than those from catalyst-equipped spark ignition engines [19,20], rising to approximately 1 g/km [19]. The soot particles are small in size (less than 0.5 pm), easily
respirable, and have carbon cores with a very large surface area on to which a
variety of organic compounds (polycyclic aromatic compounds, nitro aromatics
and quinones) are adsorbed. The number and structure of these compounds
depend on the type of engine and the mode of its operation, and may thus be
extremely variable [21]. The adsorbed compounds of diesel soot are subject to
atmospheric conversions, which make them even more toxic [22-271.
Soot particles have been shown to exhibit mutagenic and carcinogenic properties in certain cell and animal models [28-351. This is also evident from epidemiological studies concerning indoor coal burning in China [36].
I56
Effects of air pollutants
There is also growing evidence that respiratory diseases and allergic reactions
may be induced and/or enhanced by particle inhalation. Model experiments have
shown that diesel soot particles (DPs), enhanced by aqueous SO, solutions
(bisulphite), exhibit a considerable destructive potential concerning vital biomolecules such as S H compounds, polyenes, linolenic acid and certain enzymic properties [12]. The cooperative mechanism observed in the presence of DPs and
sulphite indicates monovalent oxygen reduction and subsequent lipid peroxidation
in the presence of an unsaturated fatty acid. In this process, sulphite is supposed
to function both as electron donor and radical propagating agent:
s0,2-+0,
SO3'-+ 0, + H 2 0
-+
O,'-+DP
s03'-+0,'-
(4
SO:-+
(b)
.--)
02'-+2Hf
DP'+ 0,
DP' + SO3,- -+ DP- + SO3'-
2DP'
-+
(4
(4
DP+DP-
DP'+O;-+H+
0;-+ 0;- + 2Hf
S%DDPH+02
H202+ O2
(4
As activating principles of DPs in reaction (a) both nitroaromatics and naphthoquinones have to be considered, since both classes of compounds have been shown
to undergo redox cycling, thus driving oxidative destruction in the presence of
appropriate electron donor molecules. Reactions (b) - (e) may operate as propagators of the synergistic radical chain reaction observed in the presence of both DPs
and sulphite. Superoxide plays an important role as mediator between the
DP-redox factors and different sulphur oxidation states. Disproportionation of
DPs (f) and superoxide dismutase (SOD)-catalysed dismutations [ (g) and (h)]
represent chain-terminating events, where reaction (g) would be in agreement with
the function of SOD as a superoxide-semiquinone-oxidoreductase [37].
Beside nitroaromatics and naphthoquinones iron may play an important role
as an activating principle, as is well documented for asbestos fibres [38,39].
Asbestos fibres are able to generate OH' radicals and 0,'- from H202via the
catalysis of iron as an integral part of the asbestos complex. Pulmonary epithelial
cell injury is mediated by H 2 0 2 release from asbestos-activated polymorphonuclear neutrophils (PMNs) [40]. Asbestos alone has less cytotoxic effect on
epithelial cells in leucocyte-free media; however, asbestos in combination with
PMNs causes significant damage dependent on asbestos dose. As summarized by
Mossrnan et ul. [41] several studies support 'the concept of a cause and effect
relation between activated oxygen species and the development of asbestosis'.
Therefore asbestos fibres, in addition to their mechanical properties, may act as
immobilized catalysts for Fenton- or Haber-Weiss-type reactions.
Using simple biochemical model reactions, simulating activated PMNs, the
iron-mediated formation of strong oxidants in the presence of asbestos could be
demonstrated. The free radical indicator ketomethylthiobutyrate (KMB) is
S. Hippeli and E.F. Elstner
I57
fragmented in the presence of certain strong oxidants such as the OH' radical
yielding ethylene, which can be sensitively monitored by gas chromatography.
The oxidoreductase of PMNs, responsible for the respiratory burst occurring
after activation of these phagocytes, was simulated using a diaphorase (from pig
heart), which is able to reduce molecular oxygen at the expense of N A D H . As
shown in Fig. 1 crocidolite stimulates ethylene release from KMB, triggered by the
NADH/diaphorase-system. The chelator ethylenedinitrilotetra-acetic acid
(EDTA) strongly enhances certain oxidative processes by facilitating Fe3
reduction as well as electron transfer from Fez+ to H202, thus allowing the
formation of OH' radicals according to the Haber-Weiss sequence. Addition of
EDTA to the asbestos-stimulated NADH/diaphorase system results, as expected,
in a strong increase of ethylene release. Both S O D and catalase inhibit the reaction
of the model system in the presence of asbestos and EDTA. Desferrioxamine, a
chelator forming an unreactive complex with Fe3+ ions, supresses the crocidolitestimulated ethylene release of the enzyme system. These results clearly indicate an
iron-mediated formation of reactive oxygen species via the Haber-Weiss-type
reaction.
+
+
4
Fig. 1. Ethylene release from KMB in the NADH/diaphorase system.
Reaction mixtures contained in a total volume of 2 ml: 2.5 mM KMB; 2.2 units
of diaphorase; 75 pM NADH; 400 pg crocidolite or DPs; 0.5 mM EDTA or
desferrioxamine (Desf); IO0 units of SOD or catalase; 0. I M phosphate buffer.
The reactions were done at 37 "C in the dark. Standard deviations represent
n = 6.
I58
Effects of air pollutants
As also shown in Fig. 1, crocidolite can be replaced by aqueous diesel soot
suspensions, underlining the role of iron in diesel soot toxicity.
In addition, aqueous suspensions of soot particles from domestic fuel burners
are able to function as catalysts for Fenton-type reactions (Fig. 2). In this respect,
different oxidative capacities are observed, allowing a differentiation between
diverse types of soot. Of special interest is the distinction of soot particles
stemming from mobile (traffic) and immobile (heat systems) sources respectively.
As demonstrated in Fig. 2 the soot sample derived from the chimney of a woodcharged boiler causes no increase in the ethylene formation in the N A D H /
diaphorase system.
Also, addition of EDTA shows no effect. In contrast all soot samples derived
from fuel oil (diesel soot, soot from the central heating and soot particles from
the room oil stove) enhance the NADH/diaphorase reaction and are stimulated by
EDTA. (Increasing ethylene release of the NADH/diaphorase system in the
presence of EDTA is due to ubiquitary iron impurities.) Desferrioxamine, as well
wi
U
0
2
O
W
0
+
+
.--.I
3z
4
a
IO
W
Y
v)
-
a,
m
.-a,
0
+
d
Fig. 2. The oxidative capacities of different soot types. Reaction
mixtures are as described for Fig. I. Soot (S.) samples (400 pg): diesel soot;
wood soot (from the chimney of a wood-charged boiler with an efficiency
about 730 kw); central heating soot (from the chimney of a fuel oil-fitted
boiler for central heating, efficiency 35 kw); oil stove soot (from the stovepipe of a room oil stove, efficiency 8 kw).
S. Hippeli and E.F. Elstner
I59
T
+ SOD
+ Cat.
CI
0
cn
a,
a,
ffl
6
+
.-Fig. 3. Cooperative effects of sulphite and different particle types.
Reaction mixtures were as described for Fig. I. Soot samples were as
summarized for Fig. 2.
as S O D and catalase, inhibits the ethylene release in these model systems (data not
shown).
As mentioned above, the combination of (diesel) soot particles and sulphite
exhibits a remarkably deleterious potential. In Fig. 3 the effects of sulphite on the
NADH/diaphorase system are demonstrated. Sulphite alone causes a strong
increase in KMB fragmentation. This reaction is strongly inhibited by SOD, but
not by catalase. O n the contrary this haem-enzyme stimulates the sulphitemodified ethylene release due to the NADH/diaphorase system. In the presence
of both sulphite and crocidolite, diesel soot and wood soot respectively, a comparable 2-fold enhancement of ethylene release can be observed. SOD decreases the
KMB fragmentation, whereas catalase shows no effect. In contrast the soot
samples derived from immobile fuel oil using burners inhibit the sulphitestimulated generation of reactive oxygen species due to the NADH/diaphorase
system. From this model reaction a clear differentiation between fuel oil-derived
soot samples from mobile and immobile sources appears to be possible.
In addition vital functions of human PMNs are influenced by aqueous
suspensions of soot particles in combination with sulphite in vitro ([14]; S.
Hippeli and E.F. Elstner, unpublished work).
Since both SO, and DPs are present in significant concentrations in urban air
pollution (smog) the indicated reactions may contribute to certain respiratory
disorders discussed in the context of severe air pollution.
Superoxide, the main radical generated during the interaction of sulphite and
asbestos, as well as soot particles, is shown to be also involved during influenza
infections [42]. In this connection immune defence against infectious lung diseases
I60
Effects of air pollutants
in mice is reduced after exposure t o diesel exhaust [43]. Thus superoxidegenerating systems like soot/sulphite cooperating in the alveolar space, may
modify certain immune responses in vivo.
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