carc$$0312
Carcinogenesis vol.18 no.3 pp.531–537, 1997
Estimates of the chromium(VI) reducing capacity in human body
compartments as a mechanism for attenuating its potential
toxicity and carcinogenicity
Silvio De Flora1,3, Anna Camoirano1, Maria Bagnasco1,
Carlo Bennicelli1, Gwen E.Corbett2 and Brent D.Kerger2
prior to the implementation of suitable industrial hygiene
measures.
1Institute
of Hygiene and Preventive Medicine, University of Genoa,
I-16132 Italy; and 2ChemRisk Division of McLaren/Hart, Inc., Irvine,
California 92714, USA
3To
whom correspondence should be addressed at: Institute of Hygiene and
Preventive Medicine, University of Genoa, via A. Pastore 1, I-16132 Genoa,
Italy. Telephone: 139–10–353.8500; Fax: 139–10–353.8504; E-mail:
[email protected]
Estimates of the overall reducing capacity of hexavalent
chromium(VI) in some human body compartments were
made by relating the specific reducing activity of body
fluids, cell populations or organs to their average volume,
number, or weight. Although these data do not have
absolute precision or universal applicability, they provide
a rationale for predicting and interpreting the health effects
of chromium(VI). The available evidence strongly indicates
that chromium(VI) reduction in body fluids and long-lived
non-target cells is expected to greatly attenuate its potential
toxicity and genotoxicity, to imprint a threshold character
to the carcinogenesis process, and to restrict the possible
targets of its activity. For example, the chromium(VI)
sequestering capacity of whole blood (187–234 mg per
individual) and the reducing capacity of red blood cells (at
least 93–128 mg) explain why this metal is not a systemic
toxicant, except at very high doses, and also explain its
lack of carcinogenicity at a distance from the portal of
entry into the organism. Reduction by fluids in the digestive
tract, e.g. by saliva (0.7–2.1 mg/day) and gastric juice (at
least 84–88 mg/day), and sequestration by intestinal bacteria (11–24 mg eliminated daily with feces) account for
the poor intestinal absorption of chromium(VI). The chromium(VI) escaping reduction in the digestive tract will be
detoxified in the blood of the portal vein system and then
in the liver, having an overall reducing capacity of 3300 mg.
These processes give reasons for the poor oral toxicity
of chromium(VI) and its lack of carcinogenicity when
introduced by the oral route or swallowed following reflux
from the respiratory tract. In terminal airways chromium(VI) is reduced in the epithelial lining fluid (0.9–
1.8 mg) and in pulmonary alveolar macrophages (136 mg).
The peripheral lung parenchyma has an overall reducing
capacity of 260 mg chromium(VI), with a slightly higher
specific activity as compared to the bronchial tree. Therefore, even in the respiratory tract, which is the only
consistent target of chromium(VI) carcinogenicity in
humans (lung and sinonasal cavities), there are barriers
hampering its carcinogenicity. These hurdles could be only
overwhelmed under conditions of massive exposure by
inhalation, as it occurred in certain work environments
*Abbreviations: Cr(VI), hexavalent chromium or chromium(VI); Cr(III),
trivalent chromium or chromium(III); ELF, epithelial lining fluid; IARC,
International Agency for Research on Cancer; PAM, pulmonary alveolar
macrophages; PBS, phosphate buffered saline; RBC, red blood cells.
© Oxford University Press
Introduction
At variance with trivalent chromium [Cr(III)*], hexavalent
chromium [Cr(VI)] is consistently and unequivocally genotoxic
in in vitro cellular test systems. The large majority of 436
results reported in the literature with 16 Cr(VI) compounds,
up to 1990, were positive in short-term tests evaluating genetic
and related effects (1,2). Cr(VI) is carcinogenic to humans
(3), but epidemiological studies provide evidence that its
carcinogenicity is strictly site-specific. In fact, according to
the International Agency for Research on Cancer (IARC),
there is sufficient evidence for carcinogenicity in the lung and
in the sinonasal cavity, while for other sites ‘no consistent
pattern of cancer risk has been shown among workers exposed
to chromium compounds’ (3). Review of the epidemiologic
literature and other evidence concerning Cr(VI) carcinogenicity
that has been published since the IARC review does not alter
this conclusion. As cited in the IARC monograph (3), the
epidemiologic evidence for increased risk of cancers of the
lung and sinonasal cavity is limited to conditions of high
exposure, ‘as encountered in the chromate production, chromate
pigment production and chromium plating industries’ (3),
which in the past decades involved mixed exposures to various
Cr(VI) compounds and possibly to other metals as well as
organic substances.
Experimental studies in rodents were expected to contribute
to the identification of carcinogenic Cr(VI) compounds. However, by analysing the data reviewed by IARC (3), not only
none of the six studies performed with metallic chromium or
of the 21 studies performed with Cr(III) compounds was
positive, but even less than half of the 65 studies performed
with Cr(VI) compounds (41.5%) was positive. Moreover, the
majority of positive results were generated in studies involving
certain exposure routes (subcutaneous, intramuscular, intraperitoneal, intrapleural, intrabronchial) which by-pass important
detoxification barriers and do not reproduce any human exposure (2). All patterns of experimental carcinogenicity with
Cr(VI) compounds point to the existence of threshold mechanisms, as indicated by the high frequency (58.5%) of negative
results, attributable to a complete Cr(VI) detoxification in
treated animals, or induction of cancer only under particular
conditions, e.g., (a) at extremely high doses, saturating detoxification mechanisms, (b) at implant sites only, and never at a
distance from the portal of entry into the organism, (c) in
certain targets only, selected by the lack of detoxification
mechanisms, or (d) when given in a single massive dose, and
not in the same cumulative amount fractionated into smaller
doses which can be more easily detoxified (2,4).
Therefore, Cr(VI) is potentially carcinogenic, but a series
of hurdles tend to reduce its bioavailability and to limit its
effects in the organism. Several studies published in our
531
S.De Flora et al.
laboratories since 1978 (5) have demonstrated the occurrence
of detoxification mechanisms of Cr(VI), mainly based on
reduction to Cr(III), which is not carcinogenic and does not
bear any toxicological relevance (1–3,6,7).
Based on these studies and on new experiments designed
ad hoc, we made quantitative estimates of the overall Cr(VI)reducing or sequestering capacity of some human body compartments by taking into account the specific Cr(VI) reducing
activity of body fluids, cells or organs, and their average
volume, number or weight in humans. We use the general
term sequestration when intact cells were tested and the term
reduction when cell homogenates or their subfractions were
tested in the presence of an exogenous NADPH-generating
system.
Materials and methods
The Cr(VI) reducing capacity was estimated by multiplying the specific
reducing activity of a given organ, cell population, or fluid, expressed as µg
Cr(VI) reduced per unit of weight, volume or number, by the average content
of the same organ, cell population or fluid in the human body. The Cr(VI)
reducing activity was inferred from our previous studies in the case of saliva
(8), gastric juice (8,9), epithelial lining fluid (ELF) (10), pulmonary alveolar
macrophages (PAM) (10), peripheral lung parenchyma (11) and bronchial
tree (12).
New experiments were carried out for assessing the sequestering activity
of human intestinal bacteria or of whole blood, and the reducing ability of
red blood cell (RBC) lysates or of liver homogenates. We refer to the previously
mentioned papers for technical details. Briefly, 25% liver homogenates were
prepared in 0.25 M sucrose–50 mM Tris-HC1 buffer, pH 7.4. Heparinized
venous blood was used for evaluating the Cr(VI) sequestering capacity of
whole blood. For preparing RBC lysates, blood was centrifuged at 2000 g for
15 min, washed three times with phosphate buffered saline (PBS), pH 7.4,
taken to the original volume of blood with distilled water, frozen at –80°C
and thawed three times. RBC lysates were centrifuged at 30 000 g for 15 min
in order to obtain the corresponding soluble fractions. Intestinal bacteria were
obtained from human feces by growing overnight broth cultures (10 ml) in
Oxoid nutrient broth. The bacteria were washed by centrifuging three times
the broth cultures at 10 000 g for 15 min and resuspended in PBS, pH 7.4
(1 ml). The number of viable bacteria was assessed by seeding serial dilutions
of the concentrated bacteria (1:105, 1:106, 1:107 and 1:108) in triplicate Oxoid
agar plates.
Varying amounts of sodium dichromate, dissolved in PBS, pH 7.4, at
concentrations corresponding to 0.625, 1.25, 2.5, 3.75, 5.0, 7.5 or 10 µg
Cr(VI) per vial, were mixed either with liver homogenates (6.25, 12.5, 25 or
50 µl per vial), RBC lysate soluble fractions (6.25, 12.5, 25, or 50 µl per
vial), whole blood (100 µl per vial), concentrated intestinal bacteria (100 µl
per vial), or equivalent volumes of their diluting solvents. An NADPHgenerating system (S9 mix) (13) was added to liver homogenates and RBC
lysates. After 60 min of gentle mixing at 37°C, the mixtures were evaluated
for the amount of residual Cr(VI), both by using the colorimetric method with
s-diphenylcarbazide (10) and/or by assessing mutagenicity in strains TA100
and TA102 of Salmonella typhimurium (13). Due to the turbidity of mixtures,
only the mutagenicity test system, used as a ‘biological spectrophotometer’,
could be applied in the case of liver homogenates and RBC lysates. The
amounts of residual Cr(VI), and consequently the amounts of reduced or
sequestered Cr(VI), were calculated from the regression lines relating the
initial amounts of Cr(VI) to the loss either of colorimetric reactivity or
mutagenic activity.
Results
Table I reports the volume, weight or number of selected
fluids, organs or cell populations in the human body, along
with their ability to reduce or sequester Cr(VI). Accordingly,
the overall Cr(VI) reducing or sequestering capacity of fluids,
organs or cells was calculated either on a daily basis or in
terms of saturating amounts of Cr(VI). The results of these
estimates are further summarized in Figure 1.
Saliva
Samples of saliva from five subjects (three males and two
females), incubated with Cr(VI) for 5 min at 37°C, reduced
532
1.4 6 0.2 µg Cr(VI)/ml (mean 6 SD) (8). Since the daily
secretion of saliva in humans is in the 500–1500 ml range
(14), this mechanism will reduce daily 0.7–2.1 mg Cr(VI).
Gastric juice
The Cr(VI)-reducing activity of gastric juice samples from
four fasting individuals was found in an earlier study to be
9.2 6 0.4 µg/ml fluid (8). Later on, a detailed study regarding
the reducing ability in the gastric environment was performed
in 17 individuals, both males and females, in whom samples
of gastric juice were aspirated at hourly intervals by means of
a nasogastric tube (9). The baseline of Cr(VI)-reducing activity
during interdigestive periods (mainly during the night) was
8.3 6 4.7 µg/ml, i.e. very similar to the one detected in the
previous study. After each one of the three daily meals there
were periods of 3–4 h during which peaks of 50–60 µg/ml
were recorded, averaging 31.4 6 6.7 µg Cr(VI) reduced per
ml gastric juice during these periods. It is known that in a
fasting individual the gastric secretion accounts for 1000–
1500 ml (15), and in the 4-h period after each meal an average
amount of ~800 ml is additionally secreted (16). Therefore,
the overall Cr(VI) reduction by gastric juice may be estimated
to be at least 84–88 mg per day (8.3 µg/ml31000–1500 ml
1 31.4 µg/ml3800 ml33 meals).
Cr(VI) reduction by gastric juice is due both to thermostable
components of this fluid and to food components (9). Since
gastric juice samples were centrifuged before being challenged
with Cr(VI) in order to remove gross food residues, the above
calculations underestimate the actual Cr(VI) reducing capacity
of gastric contents. It is noteworthy that the Cr(VI) reducing
activity was evaluated after 60 min of contact at 37°C with
gastric juice samples, but time-course experiments showed
that the reaction was complete within 10–20 min (9), and at
least half of it was accomplished within 1 min only
(A.Camoirano and S.De Flora, unpublished data).
Intestinal bacteria
The bacteria isolated from the feces of three male individuals
sequestered 3.8 6 1.7 µg Cr(VI)/109 bacteria. The bacterial
population in the intestine is huge, i.e. as related to 1 g of
intestinal content, 0–104 bacteria in the duodenum, 105–106 in
the ileum, and 1010–1012 in the cecum (17). The amount of
bacteria in feces mostly reflects their concentration in the
colon. The average fecal mass is 124 6 40 g/day, and its dry
mass is 21 6 5 g/day, 14–30% of which is represented by
bacteria (14). Accordingly, 2.9–6.3 g of bacteria are eliminated
daily with feces. Taking into account that 1012 bacteria weigh
~1 g and are capable of sequestering 3.8 mg Cr(VI), it can be
calculated that 11–24 mg Cr(VI) can be eliminated daily with
fecal bacteria.
Liver
Unfractionated homogenates of liver, obtained at surgery from
three males, reduced 2.2 6 0.9 mg Cr(VI)/g wet tissue, after
contact for 60 min at 37°C in the presence of S9 mix, as
evaluated by assessing the decrease of sodium dichromate
mutagenicity in strains TA100 and TA102 of S.typhimurium.
Since the average weight of the human liver is 1500 g, not
taking into account 400–800 g of blood circulating in this
organ (18), we estimated an overall reducing capacity of
3300 mg Cr(VI).
Blood
We evaluated Cr(VI) sequestration by whole blood as well as
its reduction by the soluble fraction of red blood cell (RBC)
Chromium(VI) reduction in the human body
Table I. Estimates of the overall chromium(VI) reducing capacity of organs, cell populations and fluids in the human body (see text for details)
Organ, cell population, or body
fluid
Weight of organs, number/weight/
volume of cells, or volume of body
fluids
Chromium(VI) reduction or
sequestration (mean 6 SD)
Saliva
500–1500 ml/day (4)
1.4 6 0.2 µg/ml (8)
0.7–2.1 mg/day
Gastric juice
1000–1500 ml/day
(fasting) (15)
1800 ml/meal (16)
3400–3900 ml/day (3 meals)
8.3 6 4.3 µg/ml (9)
8.3–12.5 mg/day during interdigestive
periods
125.1 mg/meal
.84–88 mg/day (3 meals)
31.4 6 6.7 µg/ml (9)
Overall chromium(VI) reducing or
sequestering capacity per individual
Intestinal bacteria
2.9–6.3 g eliminated daily with feces
(14)
3.8 6 1.7 µg/109 bacteriaa
11–24 mg eliminated daily with feces
Liver
1500 g (18)
2.2 6 0.9 mg/g liver homogenatea
3300 mg
Whole blood
4490 ml (males) (14)
3600 ml (females) (14)
52.1 6 5.9 µg/ml
intact blooda
234 mg (males)
187 mg (females)
Red blood cells (RBC)
2030 ml (males) (14)
1470 ml (females) (14)
63.4 6 8.1 µg/ml
RBC lysate soluble fractiona
128 mg (males)
93 mg (females)
Epithelial lining fluid (ELF)
37.5–75 ml (20)
23.7 6 15.9 µg/ml (10)
0.9–1.8 mg
Pulmonary alveolar
macrophages (PAM)
23 3 109 PAM (21)
4.4 6 3.9 µg/106
PAM S9 fraction (10)
136 mg
Peripheral lung parenchyma
1300 g (18)
0.2 6 0.07 mg/g
lung S12 fraction (11)
260 mg
aThis
study.
Fig. 1. Estimates of Cr(VI) sequestration or reduction by organs, cell
populations and fluids in the human body. See text for details and explanations.
lysates from three subjects, two males and one female. After
contact of sodium dichromate [100 µg Cr(VI)] with 1 ml
heparinized venous blood for 60 min at 37°C, an average of
47.9 µg was recovered from plasma, thus indicating that
52.1 6 5.9 µg had been sequestered by blood cells or reduced
by plasma itself. Similar data were obtained by measuring
residual Cr(VI) either by colorimetric analysis or in the
mutagenicity test system with strains TA100 and TA102 of
S.typhimurium. Taking into account the average volumes of
blood in males and females (see Table I), we calculated an
overall sequestration of 234 and 187 mg Cr(VI), respectively.
The RBC lysate soluble fractions from the same subjects
were assayed in the mutagenicity test system. In the presence
of S9 mix, 1 ml portions of the RBC lysates reduced 63.4 6
8.1 µg Cr(VI) within 60 min at 37°C. According to this
experimental approach and to the known volumes of RBC in
males and females (see Table I), we calculated an overall
reduction of 128 and 93 mg Cr(VI), respectively. This is an
underestimate of the overall reducing capacity of RBC, since
the RBC stromal fraction was removed by centrifugation.
These results are comparable to those obtained in a previous
study (19), in which an appreciable decrease of Cr(VI) (as
chromic acid) mutagenicity by RBC lysates was even observed
in the absence of an exogenous NADPH-generating system
(i.e. S-9 mix).
Epithelial lining fluid (ELF)
The ELF includes the surfactant and those bronchial and
bronchiolar secretions which can be recovered by bronchoalveolar lavage. Amounts of 23.7 6 15.9 µg Cr(VI) (mean 6
SD in 15 individuals, both males and females) were reduced
by each ml of ELF after contact for 4 h (10). Since the total
volume of ELF has been reported to be between 37.5 and
75 ml (20), its overall reducing capacity can be estimated to
be 0.9–1.8 mg Cr(VI) per individual.
Pulmonary alveolar macrophages (PAM)
The total number of these sweeping cells in the normal human
lung at any given time is estimated to be 23 billion, with ~50–
100 PAM per alveolus (21). We evaluated Cr(VI) reduction
by PAM S9 fractions after 30 min at 37°C. Expressed as µg
Cr(VI) reduced per 106 cells, the results were as follows: 2.4
6 1.1 (mean 6 SD in four non-smokers), 3.6 6 0.7 (eight
exsmokers), 5.8 6 1.2 (6 current smokers) and 6.3 6 1.1 (five
subjects smoking during the last 2 h before bronchoalveolar
lavage). The specific reducing activity of PAM in smokers
was significantly higher (P , 0.01) than that in non-smokers
and exsmokers. The average Cr(VI) reduction capacity was
4.4 6 3.9 µg/106 PAM in 23 subjects of both sexes (10). In
533
S.De Flora et al.
the same study we established that the reducing activity of
unfractionated PAM homogenates was 1.33 times higher than
that of the corresponding S9 fractions (10). Therefore, we
introduced this correction factor for estimating the overall
Cr(VI) reducing capacity by PAM (136 mg per individual).
Peripheral lung parenchyma
Cr(VI) reduction by S12 fractions of peripheral lung parenchyma was assessed in tissue samples taken at surgery from
71 male subjects. After incubation with sodium dichromate
for 60 min at 37°C in the presence of S9 mix, the observed
decrease in mutagenicity in strain TA100 of S.typhimurium
corresponded to reduction of 0.24 6 0.07 mg Cr(VI) per
g wet tissue (11). Interestingly, the reducing activity was
significantly higher (P , 0.05) for tissues obtained from 45
smokers (0.25 6 0.07 mg/g), as compared to 26 non-smokers
(0.22 6 0.06 mg/g), and the 19 individuals who smoked during
the last 24 h preceding surgical operation exhibited even
greater reduction capacity (0.27 6 0.06 mg/g) (11). Again, a
1.33 correction factor extrapolating from S12 fractions to
unfractionated homogenates was introduced for estimating the
overall Cr(VI) reducing capacity of the lungs (260 mg per
individual).
Bronchial tree
Peripheral lung parenchyma and bronchial tree S12 fractions
from 18 of the above mentioned subjects were compared for
their ability to decrease the mutagenicity of sodium dichromate.
There was a statistically significant correlation between the
two preparations in decreasing Cr(VI) mutagenicity, peripheral
lung parenchyma being slightly yet significantly more efficient
in accomplishing this process (12). These findings refer to
whole bronchial tissue preparations, since it was not possible
to dissect sufficient amounts of bronchial epithelium for
assessing the endpoints under study. The lack of information
on the weight of bronchial tree does not allow us to estimate
its overall reducing capacity per individual.
Discussion
The results of the present study provide for the first time
estimates aimed at quantitating the ability of organs, cell
populations and fluids in the human body to sequester or
reduce Cr(VI). It is clear that the reported data do not have
absolute precision or universal applicability for several reasons.
For example, all estimates were inferred from ex vivo experiments which, depending on the specific test design, could
overstate or underestimate the reduction or sequestration process in vivo. Inter-individual differences in these parameters
should also be taken into account to infer the reduction capacity
of various compartments in a specific person. Moreover, the
overall reducing or sequestering capacity was estimated by
hypothetically assuming a uniform distribution of Cr(VI) in
the body compartment under evaluation.
Some data, e.g. Cr(VI) reduction in saliva and gastric juice,
or its sequestration by bacteria, could be estimated on a daily
basis. All remaining data refer to the overall capacity of fluids,
organs or cells. Nevertheless, the reported estimates do not
reflect rigid saturation levels, unless following massive acute
exposure, since there is a continuous production of body fluids
and turnover of certain cell populations. For instance, 1–5
million PAM in the human lungs are removed every hour
via the mucociliatory escalator to be either expectorated or
swallowed (22). Taking into account that the mean (6 SD)
534
lifespan of human RBC is 107612 days (14), every day a
population corresponding to 1/107th of the total RBC volume,
i.e., 19 ml in males and 14 ml in females, will be renewed.
Moreover, even in organs characterized by a poor turnover of
cells, such as the liver, there is a continuous synthesis of
glutathione (GSH) and other reducing molecules which, besides
exerting protective effects locally, are exported to other organs.
As extensively discussed in previous articles (1,2,4,6,8,23–
25), Cr(VI) reduction in biological fluids (e.g. saliva, gastric
juice and ELF) and long-lived non-target cells (e.g. RBC and
PAM) is expected to greatly attenuate Cr(VI) toxicity, to
imprint a threshold character to the carcinogenesis process and
to restrict the targets of its potential activity. In fact, Cr(III),
the stable reduced form of chromium, is not mutagenic in
bacteria (1–3) and does not bear toxicological relevance in the
extracellular environment, due to its poor ability to cross cell
membranes (4). Provided Cr(VI) may overwhelm a series of
hurdles and penetrate into cells, it will be reduced to Cr(V),
Cr(IV) and Cr(III) by a complex network of mechanisms, both
enzymatic and nonenzymatic, which may also lead to the
generation of reactive oxygen species (4,26). Depending on
proximity to DNA, these processes may result in DNA damage
by these reactive species, or in trapping by cell nucleophiles
and consequently in detoxification (4). It is meaningful, in this
respect, that tissues which are typical targets for Cr(VI)
carcinogenicity in rodent assays, such as the rat skeletal muscle
after local implantation (3), have a negligible activity in
decreasing Cr(VI) mutagenicity (19). Thus, tissue-specific
susceptibility to Cr(VI) tumorigenicity correlates with intrinsically poor capacity for intracellular Cr(VI) reduction.
The investigated mechanisms and quantitative estimates on
Cr(VI) reduction may predict and explain the fate of this metal
in the human body. The massive reducing and sequestering
capacity of the blood explains why Cr(VI) exerts its toxicological consequences only at the portal of entry into the
organism, while it is not a systemic toxicant or carcinogen. In
fact, Cr(VI) carcinogenicity in humans is restricted to the
sinonasal cavity and the lung, whereas excess risks reported
for cancer at other sites were inconsistent and not convincingly
associated with Cr(VI) exposure (3). In only one out of the
65 carcinogenicity studies in rodents reported by IARC (1990),
Cr(VI) was found to be carcinogenic at a distance from the
administration site, specifically in rats receiving intramuscular
injections of lead chromate, which developed renal carcinomas
(27). However, this effect should be ascribed to lead, which
typically produces kidney tumors in rodents (28), rather than
to chromate.
In the present study, ~200 mg Cr(VI) were estimated to be
sequestered by whole human blood. About half of this figure
was found to be reduced in the presence of the soluble fraction
of RBC lysates. This difference is likely due to the fact that
preparations of RBC lysates involves elimination of their
stromal fraction by centrifugation and loss of biochemical
activities, which are partially restored by adding an exogenous
NADPH-generating system. In addition, blood plasma has
been reported to reduce up to 2 µg Cr(VI) per ml blood
(29,30). Since the mean plasma volume is 2640 ml in adult
males and 2130 ml in adult females (14), the overall Cr(VI)
reducing capacity of blood plasma can be estimated to be 4.9
and 4.2 mg, respectively. A recent investigation (31) examined
the rate and capacity of Cr(VI) reduction in human blood
in vitro and reported findings that agree well with data
generated in the present study. The major detoxification occurs
Chromium(VI) reduction in the human body
in RBCs, where since many years it is known that Cr(VI) is
selectively accumulated (32) and reduced to Cr(III) (especially
by GSH) and bound to low-molecular weight components and
chiefly to hemoglobin (33–38). These mechanisms also help
to explain why all chromium detectable in the urine of exposed
individuals is Cr(III) (3,39).
Formidable barriers hamper the potential toxicity of Cr(VI)
when introduced by the oral route or swallowed either following
reflux from the respiratory tract or following impact onto the
proximal aerodigestive tract of inhaled Cr(VI) associated with
large-size particles. After reduction in the oral cavity by saliva,
which would be sufficient to detoxify Cr(VI) contained in
several liters of drinking water at the standards recommended
in several countries (0.05–0.1 mg/l), an efficient reduction
occurs in the stomach, accounting for at least 84–88 mg per
day. A further hurdle is represented by intestinal bacteria
which, as estimated just from the aliquot of bacteria eliminated
with feces, will sequester daily 11–24 mg Cr(VI). Intestinal
bacteria are known to contain high amounts of GSH (40),
which efficiently reduces Cr(VI) (4). A Cr(VI) reducing role
has been also ascribed to enterobacterial enzymes, such as
nitroreductases (4). Since bacterial GSH is released extracellularly (40), an additional Cr(VI) reducing activity is also likely
to occur in the extracellular environment of the intestinal
lumen. All these mechanisms explain the poor absorption of
Cr(VI) in the intestine (41,42). In case some amount of Cr(VI)
may escape detoxification, it will penetrate into the blood of the
portal vein and, in case even this barrier may be overwhelmed, it
will reach the liver, which is capable of reducing grams of
Cr(VI) daily. As a consequence, Cr(VI) is poorly toxic by the
oral route. In fact, only few lethal effects were observed in
episodes of accidental ingestion of high doses of Cr(VI) by
humans (6). For instance, following acute ingestion in the
range between 8 and 20 g of Cr(VI) compounds, eight subjects
survived and three subjects died (6). The LD50 in rodents is
.50 mg Cr(VI)/kg body weight (43).
The defence mechanisms are even more effective after
repeated ingestion of smaller Cr(VI) doses, which can be
readily detoxified and, therefore, do not result in cumulative
phenomena, including mutagenicity and carcinogenicity. For
instance, a family accidentally exposed to Cr(VI) at 1 mg/l in
well water for three years was reported to have no adverse
effects (44). Dogs fed 11.2 mg/l Cr(VI) for 4 years (45), and
rats fed 25 mg/l for 1 year (46) or 134 mg/l for 6 months (47)
exhibited no adverse effects from these chronic exposures.
Similarly, a recently published investigation showed a lack
of genotoxic effects after oral administration of potassium
dichromate at up to 20 mg of Cr(VI) per liter of drinking
water, or administration of a similar amount by gavage, in
either the mouse bone marrow micronucleus assay or the rat
in vivo–in vitro rat hepatocyte DNA repair assay (48). In
addition, a three-generation study of drinking water exposure
to potassium chromate in rats failed to demonstrate treatmentrelated increases in carcinogenicity following lifetime exposures (47). This observation is in agreement with the fact that
no consistent pattern of excess risk for cancer of the digestive
tract emerged from a large number of epidemiologic studies
of Cr(VI) workers (3), despite the fact that massive exposures
by inhalation presumably involved swallowing of Cr(VI).
Furthermore, no exposure-related increase in stomach cancers
or overall cancer mortality was reported in a recent evaluation
of a residential population in China exposed for up to 6
years to Cr(VI) in drinking water derived from an aquifer
contaminated by effluent from a chromate alloy facility (49).
A series of recent studies indicate that the relatively high
reducing capacities related to oral ingestion are effective in
(a) dramatically attenuating chromium absorption into the
blood; (b) preventing any measurable entry of chromium in
the hexavalent form into the blood, (c) preventing any increase
in a putative marker for blood uptake of Cr(VI) (DNA–protein
cross-links in peripheral lymphocytes) and (d) controlling the
absorption, distribution and excretion of Cr(III) complexes
that are formed following ingestion of Cr(VI) at plausible
concentrations in drinking water (39,50–53). Each of these
studies monitored total chromium concentrations in RBCs,
plasma and urine over time to assess bioavailability and
potential Cr(VI) uptake into the blood, using RBC chromium
levels as a primary biomarker for such uptake. Human volunteers in these studies ingested water containing Cr(VI) or
Cr(III) at concentrations up to 10 mg/l for as long as 17
consecutive days. In sum, these studies suggest that ingestion
of Cr(VI) in drinking water at plausible chronic exposure
concentrations (i.e. at or below 0.5 to 2 mg/l, when yellow
discoloration becomes apparent) results in minimal uptake of
chromium into the blood. Further, the RBC and plasma
chromium concentration patterns following exposures to Cr(VI)
in drinking water at up to 10 mg/l suggest that .99.7% of the
ingested dose is reduced in the gastrointestinal tract prior to
absorption into the blood. Thus, the pharmacokinetics of
Cr(VI) at plausible chronic doses in drinking water mimics
that of organic complex forms of Cr(III) (e.g. vitamin forms
such as the tripicolinate or the glucose tolerance factor), likely
due to formation of such complexes upon contact with organic
reducing agents present in the gastric contents, tissues and
blood.
In addition to well-known non-specific defense mechanisms
of the respiratory tract (22), reduction in the ELF and in PAM
supports the view that Cr(VI) encounters efficient detoxification
barriers following inhalation. This is consistent with the
premise that even for the primary target organ of Cr(VI) in
humans there is likely a threshold for adverse effects including
cancer, that is related to the reduction capacities of intrinsic
defense mechanisms (54). The ELF layer has an average
thickness in the 0.54–1.07 µm range over the 70 m2 of alveolar
surface of each individual (20). This fluid is particularly rich
in antioxidants, such as GSH, vitamin C (ascorbic acid),
vitamin E (tocopherol), superoxide dismutase, catalase, albumin, ceruloplasmin, transferrin, lactoferrin and other proteins
(55). In rats the Cr(VI) reducing activity of ELF has been
mainly ascribed to ascorbic acid (56), but in humans GSH
levels are particularly high in ELF (57). Even more important
is phagocytosis and reduction of Cr(VI) by PAM, that we
estimated to reduce 136 mg/day/individual. The reducing
activity of rat PAM was of the same order of magnitude as
that of human PAM from non-smokers, i.e., around 2 µg
Cr(VI)/106 cells (58). Peripheral lung parenchyma showed an
appreciable Cr(VI) reducing capacity (260 mg/individual),
although it appears to be 12.7 times lower than that of the
liver, which is also known to be more efficient than the lung
in repairing DNA damage (59). It is also noteworthy that
Cr(VI) is capable of inducing its own pulmonary metabolism
(60). On the whole, there is evidence that the human respiratory
tract has considerable defence mechanisms towards Cr(VI),
although they are not as outstanding as those of the digestive
tract. These processes unequivocally support the existence of
535
S.De Flora et al.
threshold mechanisms, which could be overwhelmed only
under conditions of massive exposure by inhalation, as it
occurred in certain work environments prior to the implementation of suitable industrial hygiene measures.
In conclusion, the herein reported quantitative estimates of
Cr(VI) reduction in organs, cell populations, and fluids in the
human body provide a rationale for predicting and interpreting
the health effects of Cr(VI), including acute toxicity and longterm effects, as related to the portal of entry into the organism.
At the same time, these data may be useful to regulatory
agencies when setting Cr(VI) standards in the workplace or in
the environment.
Acknowledgements
The studies reported in this article were supported by the Italian Ministry of
University and Scientific-Technologic Research (MURST, 40% and 60%
grants).
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Received on July 31, 1996; revised on October 30, 1996; accepted on
November 12, 1996
537