10

Biological Interactions with

Mineral Particles
10.1

Interactions with Biological Systems

Once introduced into a mammalian biological system, there is no such thing

as an inert particle. Particles of obvious chemical (covalent) inertness, such as
hydrophobic talc particles, can manifest a strong physico-chemical reactivity,
in aqueous media, on account of their hydrophobic attraction for biopolymers
(e.g., proteins; cf. van Oss et a/., 1995b). hydrophilic particles such as silica
also bind proteins, by a different physico-chemical mechanism, as do many
other metal oxides (van Oss et a/., 1995a, b).
The aspecific adsorption of biopolymers, such as serum immunoglobulins,
results in the phagocytic ingestion of small particles (Absolom et a/., 1982;
van Oss 1986). Thus, inhalation by or injection of, e.g., mineral particles
in mammals, leads to attempted (if the particles are too large) or successful, phagocytic ingestion by neutrophils and/or monocytes and macrophages,
through the intermediary of serum protein adsorption, especially of immunoglobulin-G (IgG)). Adsorption of IgG, as well as of some non-IgG serum
proteins such as the most abundant serum protein, human serum albumin,
by hydrophobic particles, leads to the activation of human monocytes (monocytes are circulating phagocytic cells which are precursors of macrophages)
from peripheral blood (Nairn et al, 1998).
Thus, the introduction of small hydrophobic particles (mineral or otherwise) into mammalian systems leads to blood protein adsorption, which
in turn leads to phagocytic ingestion (when physically possible) and to the
activation of phagocytic cells. For definition and expression of the degree of

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hydrophobicity, cf. Chapter 9. Particle hydrophobicity is especially important for the adsorption and concomitant partial denaturation of the adsorbed
protein (e.g., serum albumin), which is a conditio sine qua non for the activation of phagocytic cells (Nairn et al., 1998). For the adsorption of IgG on
the other hand even adsorption onto hydrophilic, as well as onto hydrophobic
particles, can lead to phagocytic ingestion.

10.2

Polymer Adsorption

Most polymers and biopolymers readily adsorb to clay particles from aqueous
solution, see, e.g., (Theng, 1979) (general), (Parfitt and Greenland, 1970b),
for the adsorption of poly(ethylene glycol) (more usually named polyethylene
oxide) onto a Wyoming montmorillonite.
A number of authors have been interested in the adsorption of neutral polysaccharides (dextran and related polyglucosides), e.g., (Parfitt and
Greenland, 1970a; Olness and Clapp, 1973, 1975). The adsorption of neutral polysaccharides onto kaolinite as well as onto montmorillonite has been
studied (Chenu et al., 1987).
In soils and clays the most generally present biopolymers that naturally
occur in the adsorbed state on mineral and clay particles are "humic substances" , or "humic acids;" these are decomposition products of lignin, which
is the major non-cellulosic polymer in wood and other plant debris. Humic
acids (also called "allomelanins" (Merck Index, 1989)) are for the greater
part polyphenolic compounds, usually anionic polyelectrolytes, which can
complex metal ions, and are surface active and thus capable, upon adsorption onto mineral particles, to enhance their suspension stability in aqueous
media (Chheda and Grasso, 1994).

10.3

Protein Adsorption

For the interaction of clay and other mineral particles with and in biological, and especially mammalian systems, the propensity of these particles to
adsorb proteins is one of the most important factors. Hydrophobic as well
as hydrophilic clay and other mineral particles adsorb proteins from aqueous
solutions. However, the mechanisms of protein adsorption in these two cases
are entirely different.

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10.3.1

Protein adsorption onto hydrophobic surfaces

The adsorption of proteins from aqueous solutions onto hydrophobic surfaces

has a simple and straightforward physico-chemical mechanism, based upon
the global hydrophobic attraction between an apolar, or largely apolar surface
and a hydrophilic polymer (such as a protein), driven by the polar free energy
of cohesion of the surrounding water molecules; cf. Chapter 9, section 9.1.
Thus, the attraction between a protein molecule and a hydrophobic surface,
immersed in water is appropriately designated as a macroscopic interaction.
However, subsequent to the initial hydrophobic attraction between a dissolved protein molecule and a hydrophobic surface, a reorganization of the
ternary configuration is favored such that the more hydrophobic peptide
chains of the protein (which normally are sheltered inside the interior of the
dissolved protein's structure), orient themselves toward the protein's exterior, to make closer contact with the hydrophobic surface it is adsorbed to.
This reorientation of the protein molecule, occurring upon hydrophobic adsorption, is tantamount to partial denaturation and causes a reorganization
which changes the surface structure of the hydrophilic moieties of the protein molecules situated at the water interface (MacRitchie, 1972; Morrissey
and Stromberg, 1974). The structural changes brought about by hydrophobic adsorption give rise to enhanced reactivity with human phagocytic cells,
whereas protein adsorption onto hydrophilic surfaces does not (Nairn et al.,
1998). Nonetheless, a certain, but lesser degree of structural change (especially in the secondary conformation) upon protein adsorption onto hydrophilic mineral surfaces also can occur, cf. (Haynes and Norde, 1995; Buys
et al., 1996; Maste et al., 1997; Sheller et al, 1998). With time a small proportion of hydrophobically adsorbed protein becomes virtually irreversibly
attached, due to hysteresis (Sheller et al., 1998; van Oss et al., 2001a).

10.3.2

Protein adsorption onto hydrophilic surfaces

Upon surface-thermodynamic analysis of protein adsorption onto hydrophilic

surfaces such as silica or glass, based on the known surface properties of the
hydrophilic protein as well as of the hydrophilic mineral substratum, one
would arrive at the conclusion that the macroscopic-scale interactions in a
neutral aqueous medium are so strongly repulsive that adsorption should
not occur, cf. van Oss et al. (1995a). In spite of this prediction, protein
adsorption onto hydrophilic glass, silica, etc. does take place, at neutral pH
(MacRitchie, 1972; van Oss et al., 1995b). The mechanism of protein adsorption onto hydrophilic surfaces is quite different from that operative with

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hydrophobia surfaces. Whereas the latter is due to a global, macroscopic

scale attraction over the entire surface, hydrophilic adsorption is a microscopic scale attraction of the protein molecules onto tiny discrete cationic
sites that are dispersedly distributed throughout the overall anionic mineral
surface (van Oss et al., 1995a). Experimental confirmation of the discrete
site plurivalent metal ionic attraction for an anionic peptide moiety of the
protein is furnished by the observation that the adsorbed protein can be displaced from its site of attachment by complexing agents which specifically
bind to plurivalent metal ions, such as Na2EDTA (van Oss et al., 1995b),
or sodium hexametaphosphate. A metal ion site, embedded in an overall
hydrophilic anionic surface causes its immediate vicinity to be hydrophobic. Thus, the microscopic scale attraction between a hydrophobic metal
ion site and the hydrophilic anionic peptide moiety of a protein molecule, is
due to both an electrostatic and a hydrophobic attraction. Nonetheless, in
the interaction between a dissolved protein and a metal oxide surface, the
overall (macroscopic) repulsion dominates roughly three to five-fold over the
local discrete-site (microscopic) specific attraction. This is readily demonstrated by measurement of the kinetic adsorption rate constant of, e.g., the
adsorption of human serum albumin onto silica surfaces. Comparing both
the overall macroscopic repulsion energies, averaged over all distances and
all orientations (leading to net repulsion) of the protein molecules, with the
microscopic attraction energies, averaged over all distances and all favorable
orientations (leading to net attraction) of the protein molecules, showed that
a net average repulsion energy, AG.^. ~ +6.91 kT prevailed between one
protein molecule, i, and a discrete silica surface site, j, immersed in water,
w, (where 1 kT = 4.045 x 10"21 Joules, at 20C). Following von Smoluchowski's theoretical analysis (1917) of this type of encounter, the ratio of
improbability to probability (f) of the encounter of an albumin molecule with
a silica surface was of the order of f = exp(-6.91) = 10~3, corresponding to a
kinetic association (rate) constant ka 7.25 x 104 L mol"1 sec"1 (Docoslis
et al., 2000a). This does not at all mean that practically no albumin would
ultimately adsorb onto silica. Given that the kinetic dissociation constant
was found to be k^ = 3.41 x 10~3 sec"1, the equilibrium adsorption constant,
Keq Tk a /k d = 2.13 x 10~7 L mol"1. this value was only slightly smaller
than the Keq found from Langmuir isotherm distributions. It gives rise to
a respectable free energy of adsorption, AG. . = Tn(2.13 x 107 x 55.56)
= -20.9 kT. It should be noted that as Keq k a /ki, and as both Tka and
kd are proportional with von Smoluchowski's improbability/probability factor, f, Keq is independent of these improbability/probability considerations
which pertain only to the kinetics of adsorption and not to its final outcome.

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With time, a proportion of the hydrophilically adsorbed protein also becomes

practically irreversibly attached, due to hysteresis (van Oss et al., 2001 a).

10.3.3

In vivo consequences of protein adsorption onto

clay and mineral particles

The in vivo effects of protein adsorption onto solid surfaces are hard to follow,
because of the Vroman effect (Bamford et al, 1992). The Vroman effect is
the phenomenon whereby, when solid surfaces are exposed to mixtures of
blood plasma proteins, such surfaces adsorb different proteins sequentially,
so that with time, first one, then another, and then a third protein, etc.,
finds itself predominantly adsorbed
A few salient consequences of the adsorption of, inter alia, plasma proteins are outlined here, with a view to their connection with the pulmonary
pathogenesis of certain asbestos and other clay and mineral particles, discussed in the following section.
Adsorption of the three most abundant plasma proteins (albumin, fibrinogen, IgG) onto hydrophilic surfaces has no apparent effect on monocyte
activation, whereas the adsorption of the same plasma proteins onto an otherwise inert but strongly hydrophobic (e.g., silicone) surface, strongly activates
these phagocytic cells (Nairn et al., 1998).
Adsorption of the immunoglobulin, IgG, onto hydrophilic as well as onto
hydrophobic particles causes such particles, coated with (human) IgG, to be
ingested by phagocytic cells as a consequence of the fact that phagocytic
cells have surface receptors for the Fc moieties of the subclasses, IgGl and
IgG3, of IgG (Absolom et al., 1982).
Needle-shaped mineral particles such as the amphibole asbestos fibers
(crocidolite, amosite, tremolite) and the fibrous zeolite, erionite, having adsorbed IgG, but which have a low dissolution rate under intra-phagosomal
liquid conditions, are continuously, but largely unsuccessfully, attacked by
phagocytic cells, often for periods of years and even decades (van Oss et al.,
1999). This causes the fibers to become coated with a "ferruginous" layer
consisting of iron-containing proteins (e.g., ferric hemosiderin), as residues
of the iron-containing oxidases and peroxidases which are produce by phagocytic cells with a view to the oxidative destruction of ingested particles, such
as microbes (van Oss et al., 1999). Whether the thick ferruginous layers
around fibrous mineral particles, embedded in the lung or in the pleural
cavity, often during decades, are harmful or, on the contrary, whether they
lend a long-term (although in many cases not indefinite) protection against

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pathological sequelae, is not known (van Oss et al., 1999).

10.4

Pulmonary Pathogenesis

10.4.1

Small, roughly spherical particles

In the normal course of events, the occasional inhalation of a few small (no dimensions much greater than a few /^m) clay or other mineral particles causes
no harm, as such particles become quickly engulfed by phagocytic cells in
the lung (mainly macrophages) and are dissolved by the phagosomes within
the phagocytes. Even when the rate of dissolution is exceedingly slow, as in,
e.g., hydrophobic talc particles, they are ultimately removed by phagocytemediated transport, ending usually in gradual elimination through the upper
respiratory system (Singer et al., 1972). The main problem with the inhalation of small particles (as defined above) arises when, e.g., workers or miners
are continuously exposed to such particles, over long periods of time. In such
cases phagocytic removal cannot keep up with the continuous re-supply and
the particle overload can then ultimately cause chronic pulmonary pathology. Silicosis is one example of such an outcome. It should be remarked
that, other factors being equal, hydrophobic particles are more dangerous
than hydrophilic particles. This is because they bind more protein, they
activate leukocytes more strongly and they dissolve more slowly intraphagosomally (van Oss et al., 1999). Now, usually silica particles are, rightly,
considered to be hydrophilic. However, when freshly ground, or when particles have otherwise been broken or have undergone diminution, silica and
most other mineral particles become hydrophobic (Wu et al., 1996). It was
found by Vallyathan et al. (1995) that the inhalation of freshly silica leads to
increased lung inflammation. Talc particles are among the most hydrophobic
ones (Giese et al., 1996) and its continuous use involving mucous surfaces and
particularly when inhaled (e.g., by babies), or used intravenously, can give
rise to severe respiratory disability (Hollinger, 1990; van Oss et al., 1999).

10.5

Needle-shaped or Fibrous Particles

10.5.1

The most dangerous fibrous particles

Clay and other mineral particles that are needle-shaped or fibrous, with a
long-axis length that is significantly greater than about 15 /xm will,upon inhalation, elicit engulfment by phagocytic leukocytes, which are however too

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small to engulf them completely. Thus, needle-shaped or fibrous particles

tend to remain embedded in the lung tissue, because phagocytes are unable
to transport them elsewhere. Scanning electron micrographs of lung tissue
obtained from former asbestos workers upon autopsy clearly show the continuing but vain attempts by macrophages to phagocytize the needle-shaped
fibers; the coating with Fe-containing protein is also visible on the clean fiber
surface which is not covered by macrophages (Sorling and Langer, 1993; van
Oss et ai, 1999). Those needle-shaped particles that have, in addition, an
extremely slow dissolution rate under intra-phagosomal conditions cannot be
slowly digested bit-by-bit, so that these needles tend to remain in the lungs
for decades and ultimately cause neoplasms. In this category are the amphibole asbestos varieties, crocidolite (blue asbestos), amosite (brown asbestos),
tremolite, as well as the zeolite, erionite. The needles of all of these are rather
rigid and sharp so that they, once inhaled, tend to pierce the lung tissue and
congregate in the pleural sac where, after one or more decades pleural cancer (malignant mesothelioma) ensues. The lag period is typically 20 to 30
years after first exposure to erionite (Baris, 1987) and on an average about
30 years but in a few cases more than 50 years, after exposure to crocidolite,
cf. Kane (1993). Malignant mesothelioma is quite rare, but almost invariably fatal. When the disease occurs in someone who earlier in life had been
exposed to an amphibole asbestos variety (usually crocidolite), or to erionite,
that exposure may be held to have been the cause of the mesothelioma, even
when the exposure had been relatively brief, e.g., a year or less (van Oss et
a/., 1999). This particular outcome of exposure to asbestos appears to have
no connection with whether or not the patient had been a tobacco smoker
(Kane, 1993).

10.5.2

The less dangerous fibrous (asbestos) particles

In this category belongs mainly the white phyllosilicate asbestos (chrysotile).

Now, the rate of dissolution of chrysotile fibers is about an order of magnitude faster than that of the amphiboles and of erionite (van Oss et a/., 1999).
Chrysotile thus should disappear fairly quickly after inhalation, which is confirmed by autopsy studies (Roggli, 1990). Contrary to amphibole and erionite exposure (which ultimately mainly leads to malignant mesothelioma, see
above), long-term chrysotile exposure can lead to lung cancer (Kane, 1993),
especially among smokers (Selikoff et al., 1964). Chrysotile asbestos is by far
the most common variety used, e.g., in the construction industry. There is
little doubt that occasional exposure to chrysotile is not dangerous (phagocytic cells clear away the fibers fairly quickly, thanks to their relatively quick

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solubility rate). However, for long-term workers with chrysotile, who undergo
continuous exposure to the fibers, thus constantly replenishing the material
faster than their phagocytes can break it down, exposure to chrysotile remains dangerous, especially if they are smokers, which according to Selikoff
et al.(l9Q4) increases the danger 50-fold. It appears therefore much safer
(and cheaper) not to remove chrysotile asbestos from buildings, but rather
to encapsulate the material with a plastic resin, to safeguard the occupants
from even minor exposure, and the asbestos removers from further danger.
It is of course still essential to perform a prior analysis of the asbestos in
question to ensure that it consists of only chrysotile. This is because some of
the chrysotile mined, e.g., in Canada, may contain a small but non-negligible
proportion of the amphibole tremolite, which is dangerous, even after relatively brief exposure. For the lack of danger of occasional brief exposure to
chrysotile, see also Moss (1995).
The reason why needle-shaped amphibole asbestos fibers typically penetrate into the pleural cavity, whereas chrysotile fibers do not, lies in their
different morphology and pliability. Microscopic examination reveals that
amphibole asbestos fibers are indeed needle-like, rigid for their small diameter (typically only a few /mi), and straight. Chrysotile fibers on the other
hand are somewhat thinner, often longer, frequently curved, and even somewhat curly, which makes then less capable of piercing living tissue, so that
they are more prone to remain in situ, until digested.

10.5.3

A few proposed physical or chemical correlations with the pathogenicity of, e.g., amphiboles that turn out to be erroneous

Judging by the differences in iron-contents of crocidolite and chrysotile,

Hardy and Aust (1995) proposed that the high iron content of the amphibole
asbestos fiber, crocidolite, was the cause of its much greater carcinogenicity
than the chrysotile fiber, which, indeed, contains forty times less iron than
crocidolite or amosite. However, the fibrous zeolite erionite, also contains
much less iron (about 18 times less iron than the amphiboles, and is also
much less tightly bound; cf. van Oss et a/.(1999). As the mechanisms of pulmonary carcinogenesis of amphibole asbestos needles and of erionite needles
are remarkably similar, the differences in the iron contents of amphibole and
chrysotile asbestos species appear to be irrelevant.
Another apparent difference between chrysotile and crocidolite has been
proposed to lie in their electrical surface () potential (Light and Wei, 1977a,

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b). In these studies signs of the ^-potentials of two chrysotile samples (of
unknown geographic origin) turned out to be positive, whilst the signs of
the ^-potentials of the amphiboles, crocidolite, amosite and anthophyllite
were negative. Now, positively charged mineral particles are, on the whole,
fairly rare (Giese et al., 1996). In another study (van Oss et al., 1999),
upon electrophoretic analysis of six chrysotile samples, it was found however
that three different samples (two from Quebec, one from Ontario, Canada)
were indeed positively charged, but three other chrysotile samples (two from
Arizona, one from Zimbabwe) were negatively charged. The amphiboles that
were tested (two crocidolites and one amosite sample) were also all negatively
charged. Thus the sign of charge of asbestos particles does not correlate with
the pathogenicity of the species.

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