Toxicology

Letters
ELSEVIER Toxicology Letters 82183 (1995) 287-293

A new mechanism for the toxicity of ozone

William A. Pryor”,“, Giuseppe L. Squadrito”, Mitchell Friedmanb

“Biodynamics Institute, Louisiana State University, 711 Choppin. Baton Rouge, LA 70803-1800. USA
‘Deptartment of Medicine. Tulane University Medical Center, New Orfeans, LA 70112-2699. USA

Abstract

Ozone, with its high reactivity, is entirely consumed as it passes through the first layer of tissue it contacts at the
lung/air interface. This layer includes the epithelial cell lining fluid (ELF) and, where the ELF is thin or absent, the
membranes of the epithelial cells that line the airways. Thus the biochemical changes that follow the inhalation of
ozone must be relayed into deeper tissue strata by a cascade of ozonation products. Lipid ozonation products
(LOP) are suggested to be the most likely relay molecules of ozone’s signal. This is because unsaturated fatty acids
are present in relatively high concentrations in both the ELF and in pulmonary cell bilayers, and ozone reacts with
unsaturated fatty acids to produce ozone-specific products. Further, LOP are finite in number, have structures that
are predictable from the Criegee ozonation mechanism, and are small, diffusible. stable (or meta-stable) molecules,
similar to other lipid-derived signal transduction species. Preliminary data show that individual LOP cause the
activation of specific lipases, which trigger the release of endogenous mediators of inflammation.

Keywords: Ozone; Lipid ozonation product; Inflammation: Epithelial cell lining fluid; Lipase; Phospholipase

1. Introduction cascade of lipid ozonation products (LOP) rather

The inflammatory effects of ozone have been
demonstrated in both animal and human studies
[l]; effects include airway hyperactivity, in-
creased epithelial macromolecular permeability,
neutrophil infiltration, and airway mucus hyper-
secretion [l-6]. There is strong evidence for the
presence of lung inflammation several hours after
exposure [l]. Despite the long history of the
study of ozone, the molecular steps that cause
this pathology are not known.
We here review the suggestion that the toxic
effects of ozone are due, at least in part, to a
* Corresponding author, Tel.: 504 388 2063; Fax: 504 388
4936: e-mail: Bill.Pryor@Chemgate.Chem.LSU.Edu.
than to ozone itself [7,8]. Fig. 1 is a cartoon
showing the proposed cascade mechanism.
The cascade mechanism originated in calcula-
tions that suggest that ozone is too reactive to
penetrate far into tissue; only a very small
fraction of the total dose of ozone can pass
unreacted through a bilayer membrane, and
none can pass through a cell [9]. Yet we know
there are both pulmonary and extra-pulmonary
effects of ozone, and so we must ask: how do
these effects arise?
It is logical to propose that a cascade of
ozonation products is responsible for the damage
that occurs when ozone is inhaled. Ozone must
react in the first layer of tissue it contacts at the
lung/air interface. This layer includes the epi-

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288 W.A. Pryor et al. I Toxicology Letters 82183 (1995) 287-293

must be recognized that the cascade mechanism

is a ‘Tinker-to-Evers-to-Chance’ relay of a signal,
air tissue boundary and, as in baseball, the nature of the shortstop
Thin stratum
where 03reacts
and second baseman is critical in determining the
with unsaturated speed of the relay and the damage done. And so
fatty acids
we must ask: what is the nature of the products
that relay the effects of ozone?
For several reasons, we believe that LOP are
Lipase activation
(& possibly other effects)
the most likely transmitters of ozone’s message.
Firstly, the ELF is about 90% lipid and 10%
J 1 \
Release of endogenous mediators
protein, and the concentration of unsaturated
fatty acids, and particularly mono-unsaturated
of inflammation (e.g., PAF)
fatty acids, in the lipids is appreciable. Table 1
Fig. 1. A cartoon showing the cascade mechanism for ozone lists the types of lipid that occur in human ELF:
toxicity. Ozone reacts with lipids to form LOP: these LOP as can be seen, phosphatidylcholine is the princi-
then activate lipases. and perhaps have other biological
pal lipid. Table 2 gives the percent of unsatu-
properties. The activated lipases result m the production and
release of endogenous cellular signal transduction molecules
rated fatty acids present in human ELF; phos-
such as eicosanoids. PAF, and others. phatidylcholine contains about 7% palmitoleic
and about 15% oleic, plus small amounts of more
thelial cell lining fluid (ELF) and, where the ELF highly unsaturated fatty acids.
is thin or absent, the membranes of the epithelial Secondly, some ozone is destroyed by reaction
cells themselves that line the airways. But, it with antioxidants such as vitamin E, ascorbate,

Table 1
Major phosphohpids of human ELF and lung tissue

Phospholipid” ELFh (%) ELF’ (%) ELFd (%) Lung tissue’ (% )

Phosphatidylchohne 73 69 68 81
Phosphatidylglycerol 12 11 10 9
Phosphatidylethanolamine 3 8 5 2

The percentage composition refers to the surfactant isolated from minced lung tissue by repetitive centrifugatton.
11The phospholipid fraction typtcally accounts for IO-90% of the total lipid [34].
’ Ref. [35].
’ Ref. [36].
’ Ref. [37].
’ Ref. [38].

Table 2
Percentage unsaturated fatty acids present in human ELF major phospholipids

Fatty acid PC” PE PC

CH,(CH,),CH=CH(CH,),C02H
Palmitoleic acid 8,5 4 5
CH,(CH>),CH=CH(CH,),CO,H
Oletc acid 17,ll 47 34
CH,(CH,),CH=CHCH2CH=CH(CHL),C0,H
Linoleic acid I,4 8 3
CH,(CH,),CH=CHCH$H=CHCH$H=CHCH$H=CH(CH~),C02H
Arachidonic acid 0.2 5 1
a The first entry in this column is calculated from Ref. [36] and the second is from Ref. [35]. All other values are from Ref. [36].
 W.A. Pr_vor et al. I Toxtcology Letters X3X.3 (1995) 2X7-29_? 789

and glutathione, but these reactions probably are
part of a sacrificial protection system that leads
to few toxic products. (They may, however, lead
to signals of generalized oxidative stress such as
depleted glutathione [lo].) Furthermore, despite
this protective screen of antioxidants, some
ozone does react with lipids in the lung [8,11-161.
Thirdly, lipids give small, diffusible products
upon ozonation, rather than the less defined
products formed from proteins [17]. LOP are
stable (or meta-stable) molecules with structures
similar to known lipid-derived signal transduc-
tion species.
The ozonation of lipids gives products with
structures that are predictable from the Criegee
mechanism of ozonation; this mechanism is
shown in Fig. 2. If ozonation occurs in a partly
aqueous area, such as the ELF, then the ozona-
tion process produces aldehydes, hydroxy-
hydroperoxides and small amounts of the
Criegee ozonide.
The relative amounts of the products that are
formed from the ozonation of lipids can be
predicted from the known rate constants with
which the double bond in the lipid reacts with
ozone [l&-21] and the relative amounts of the
H o-o
&+
unsaturated fatty acids in the ELF and pulmon-
ary membranes. Since, as we have seen, only a
limited number of mono-unsaturated fatty acids
(MUFA) and polyunsaturated fatty acids
(PUFA) occur in lung tissue, only a limited
number of LOP will be formed.
For example, the most prevalent MUFA in the
ELF are palmitoleic (16:l(n - 7)) and oleic acids
(18:l(n - 9)), which give heptanal and nonanal as
their aldehydic products upon ozonation.
Furthermore, these aldehydic products are rela-
tively specific to ozonation, since MUFA do not
undergo autoxidation (a process that also can
produce aldehydes). Fig. 3 outlines possible LOP
from various types of pulmonary lipid.
2. Structures of the LOP
Note that ozonation of an olefin in the pres-
ence of water can give rise to either the Criegee
ozonide or to the fragmentation of the substrate
into 2 different pairs of aldehydes and hydroxy-
hydroperoxides [22]. For example, if the olefin is
a lipid such as l-palmitoyl-2-oleoyl-sn-glycerol-3-
phosphatidylcholine (POPC), then the LOP
shown in Fig. 4 are formed. These LOP include
the POPC Criegee ozonide (POPC-Oz) and 2
aldehyde-hydroxyhydroperoxide pairs, one in
n
which the aldehyde is attached to the glycerol

--c megee Oronlde backbone (PC-Ald) paired with a 9-carbon hy-

0,
droxyhydroperoxide (HHP-C9), and where the
WJ R-C
Jo R'-C/”
\‘ OH hydroxyhydroperoxide is attached to the glycerol
\ +
H oon backbone (PC-HHP) and a 9-carbon aldehyde
AIdehyde Hydroryhydroperoxlde
(Ald-C9; nonanal) is released. Thus, 5 principal
LOP are formed from POPC (if stereoiosomers
RI-C
/p are ignored). As can be seen from this example,
+ H202
\
n the number of LOP that are formed from the
ozonation of pulmonary lipids is a finite number
‘c=d + 0, + Hz0 - R-C
4” + R-C
/p + WI, of species that can be synthesized and tested
/ \ \ \
R R’ H H both in vitro and in vivo for biological effects.
Fig. 2. The top portion of this figure shows the mechanism of
ozonatton of an olefin (such as an unsaturated fatty actd) that 3. Biological effects of LOP
contams a CIS double hond. Ozonation can produce either the
Crtegee ozonide or. if water is present. a molecule of Airway epithelial cells respond to stimulation
aldehyde and one of a HHP. The HHP is in equilibrium wtth
with the release of a variety of pro-inflammatory
another molecule of aldehyde and hydrogen peroxide. The
net reaction. therefore. gives 2 moles of aldehyde and 1 mole
lipid mediators such as eicosanoids [23,24] and
of hydrogen peroxide per mole of ozone and olefin used PAF [25]; reactive oxygen species [26,27]; and
p9.401 cytokines [28,29]. Synthesis and release of many
290 W.A. Pryor et al. I Toxicology
Compound Class Lipid Precursor
I. Aldehydes Palmitoleic-containing
Lipid
Oleic-containing
Lipid
1-Palmitoyl-2-(palmitoleoyl,
oleoyl or linoleoyl)-sn-glycero-
3-phosphocholine
II. Hydroxyhydroperoxides Palmitoleic-containing
Lipid
Oleic-containing
Lipid
l-Palmitoyl-2-(palmitoleoyl,
HOO(HO)HC(CH2)$02+H
oleoyl or linoleoyl)-sn-glycero-
3-phosphocholine
Ill. Criegee Ozonide 1-Palmitoyl-2-Oleoyl-
sn-glycero-3-phospho
choline
CH3(CH2)7H60\CH(CH&C02+H
Fig. 3. Structures and their acronyms for LOP that are formed when various types of pulmonary lipids undergo ozonation.
Fig. 4. The LOP formed when POPC undergoes ozonation.
POPC is an important unsaturated lipid in pulmonary tissue,
so the LOP shown are possible relay molecules for the
toxicity of ozone into deeper tissue strata than ozone itself
can penetrate.
of these substances is stimulated by ozone expo-
sure [25]. The in vitro exposure of epithelial cells
to ozone results in dose-dependent increases in
Letters 82183 (1995) 287-293
Structure
Abbreviated Name
CH3(CH2)&H0 Ald-C7
CH3(CH2),CH0 AIQCS
CH~(CH~)I&O~;CH~ PC-Ald
0HC(CHM02CH
I
CH20P(0)3(CH2)2N(CH3)3
CH3(CH2)$H(OH)OOH HHP-C7
CH3(CH2)7CH(OH)OOH HHP-C9
CH~(CH~)I&O~CH~
PC-HHP
CH2OP(O),(CHM(CH,)3
CHG-UMCO~CH~
POPC-02
‘Od
CH2OP(OMCHM(CH3)3
PLA,, PLC, and PLD activity [25]. The activa-
tion of PLC in epithelial cells appears to be
G-protein dependent [30]. This suggests that
airway epithelial cells can respond to stimuli
through complicated signal transduction path-
ways that involve specific receptors that link G-
proteins, PLA,, PLC, and PLD. We therefore
have tested whether LOP are the signal mole-
cules that are responsible for these effects.
Our preliminary data show that individual
LOP activate specific lipases both in vitro in a
liposomal system [31] and in cells in culture [32].
Thus, LOP do appear to produce some of the
same effects that have been observed for, and
attributed to, ozone itself.
4. Cell culture data
Human bronchial epithelial (BEAS 2B) cells
were incubated for 60 min with the LOP shown
 W.A. Pryor et al. I Tox~ology Letters 82183 (1995) 287-293 291

Table 3
Polarized release of AA from BEAS 2B cells exposed to LOP (percent change from control) [32]

Measured effect AldC7 HHP-C9 PC-Ald POPC-oz

Apical AA release (percent of control) 9421 P=NS 9O-cl P=NS 39127 P<O.Ol 314%7P<O.O1

Table 4
The effects of LOP on PLC-mediated IP accumulation in BEAS 2B cells (percent change from control) [32]

LOP PC-HHP HHP-C9 PC-Ald POPC-oz

IP, (percent control) 14551 P<O.Ol 139% 1 P<O.Ol 74?1 P=NS 7351 P=NS

in Fig. 3, at 10 PM concentration. The media 5. Membrane effects of LOP

from the apical compartments were collected and
arachidonic acid (AA) release was measured. In a liposomal model membrane system, and
The results are shown in Table 3 as percent of with POPC as the lipid, we have found that
control. There was a significant increase in PLA, recognizes and hydrolyzes the POPC-Oz
[3H]AA release after incubation with either the with a rate comparable to AA [31]. This could
ozonide of POPC (POPC-Oz) or the derived represent a defense mechanism against ozone,
aldehyde (PC-Ald). Compared to control and at since this process would clear ozonides from cell
a concentration (10 PM) that is lo-fold lower membranes. However, this process also liberates
than other reported effects of similar LOP [12]. a fatty acid ozonide, which may have biological
Neither the Ald-C9 nor HHP-C9 caused a signifi- effects.
cant release. Furthermore, these results are simi- In the same system, PC-Ald is not recognized
lar to those we obtained during exposure to by PLA,; however, when PC-Ald is incorporated
ozone itself [33]. In that study, BEAS 2B cells into a liposomal membrane, the rate of hydrol-
were prelabelled with [“H]AA and then stimu- ysis of unaltered fatty acid PC by PLA, is
lated with melittin (2 pg/ml) and [“H]AA re- increased. We suggest that this results from an
lease occurred predominantly into the apical order-disrupting effect that is produced when
compartment. However, when the cells were damaged and more hydrophilic LOP are incorpo-
exposed to ozone, the predominant [3H]AA rated into a membrane [31].
release occurred in the basolateral compartment.
Both the ozone-induced and HHP-C7-induced Acknowledgements
polarized release of [3H]AA appears to be due
to PLAz activation since no PLC activation was This work was supported by grants to WAP
subsequently found. from the Health Effects Institute and the Nation-
To study the effects of LOP on PLC-mediated al Institutes of Health and to MF from the
inositol phosphate (IP) accumulation, BEAS 2B Environmental Protection Agency.
cells were incubated with [3H]myo-inositol (1
&i/ml) for 72 h and then exposed to LOP (10 References
PM each), using similar controls as above (see
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