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Begin 04/20/10
In a 2009 review of human carcinogens by the International Agency for Research on Cancer
(IARC) arsenic, beryllium, cadmium, chromium, and nickel, are categorized as Group 1
carcinogens: confirmed human carcinogens. As of 2008 the IARC classified Co in Group 2B,
possibly carcinogenic to humans.
[slide 1]
The chemistry of metals is complex, but there are some general observations that can be made
about mechanisms of carcinogenicity. All of the carcinogenic metals are associated with the
generation of ROS, either directly through redox chemistry or indirectly be interacting with
antioxidant defenses, for example depletion of GSH. The carcinogenic metals also appear to
affect cell signaling systems and are involved in activation or suppression of genes that can be
linked in one way or another to cell transformation. Cr is distinct in that, in addition to acting
through ROS and cell signaling pathways, it directly reacts with DNA, resulting in
characterizable lesions that are promutagenic and could also lead to cell transformation.
Considerable work has been published on Cr and As. Because of the time constraint, I selected
Cr and Cd to discuss in detail. I selected Cr because it is distinctive in its reactivity with DNA
and Cd, because it is of interest to us at UNC by virtue of being the object of our Superfund
research grant. We will look at Cd first.
Cadmium is relatively insoluble in all its forms, so availability and uptake are relevant to
describing the activity of Cd, in contrast to organic carcinogens that we have discussed.
Cadmium absorption shows marked route dependency with only ~5% of an oral dose absorbed
by the gastrointestinal tract. Cadmium absorption from the lung is very high, with upwards of
90% of a dose being absorbed. Cd lies below Zn in the periodic table so like Zn, is a transition
metal having its outer d-shell completely filled. Like Zn, Cd is biologically active in the 2+
valence state and as a consequence, Cd mimics Zn and can replace Zn in biological complexes,
particularly Zn finger proteins. Under certain circumstances, Cd can also replace Ca2+, and this
also has implications in its carcinogenic activity as we shall see. There is a common pathway for
absorption of cadmium with iron through the divalent metal transporter-1 protein (DMT-1)
which enhances uptake of cadmium during iron deficiency.
The major mechanisms involved in Cd carcinogenesis can be broadly categorized into four
groups (next slide), aberrant gene expression, inhibition of DNA damage repair, inhibition of
apoptosis, and induction of oxidative stress, and all of these pathways are interconnected.
[slide 2]
Aberrant gene expression
Recent developments in gene expression studies, especially those in toxicogenomics, have
facilitated the identification of a large number of genes and provide insight into mechanisms that
are potentially involved in Cd carcinogenesis. The sub-group of genes whose expression is
influenced by Cd exposure and which may also be involved in Cd toxicity and carcinogenesis
can be organized into five categories:
[slide 3]
1. Immediate early response genes, abbreviated IEGs, 2. Stress response genes, 3. transcription
factors, 4. translation factors, and 5. miscellaneous genes. There is overlap between the stress
response genes, transcription factors and IEGs.
Immediate early response genes (IEGs)
The group of genes called immediate early response genes (IEGs) are protooncogenes that
undergo early transcriptional activation in response to mitogenic stimuli, and they are
overexpressed in response to Cd exposure. In this group are some familiar names: c-fos, c-jun
and c-myc which are transcription factors. The connection between overexpression of the early
response genes and carcinogenic potential of Cd lies in the observation that IEGs are frequently
found overexpressed in tumors and in cells undergoing proliferation. The Cd-induced
overexpression of the IEGs can be transitional, lasting for a few hours, or sustained, such as in
the case of cells that are transformed by exposure to Cd.
Effects on the levels of secondary messengers such as ROS and Ca2+ have been suggested as the
mechanism of Cd-induced overexpression of the immediate early response genes. Elevated
cellular levels of Ca2+ have been associated with overexpression of the transcription factors c-fos,
c-jun and c-myc in cell culture. Remember that the jun-fos complex is a bZIP heterodimer that
comprises the transcription factor AP-1, which responds to multiple stimuli, including stress and
growth factors initiating cell division. The c-myc gene codes for a bHLH transcription factor
which is involved in activation of a large number of diverse proteins (15% of all genes). The role
if Ca2+ in activation of IEGs is postulated to be interaction with response elements such as the
serum response element (SRE) or cAMP-response element binding protein (CREB) that are
present in the promoter or enhancer regions of the IEGs. In a second Ca ion-dependent pathway,
elevated Ca2+ levels can trigger specific kinases which in turn can catalyze the phosphorylation
of transcription factors resulting activation and consequent the deregulation of their target genes.
One specific example is the activation of protein kinase C (PKC) which has been demonstrated
in the overexpression of c-fos and c-jun in response to Cd exposure.
Stress-response genes
The expression of a group of genes collectively referred to as stress response genes is induced in
order to combat the stress caused by exposure to Cd. Among the stress response genes which are
induced are genes involved in the synthesis of metallothionein (MT), heat shock proteins and the
oxidative stress response protein glutathione (GSH). Earlier in the course, we used the MT gene
as an example of how response elements function. The MT protein is a low molecular weight
protein containing 30% cysteine, which chelates metals because of its high sulfhydryl content,
and thus serves as a defense against metal toxicity.
GSH is the most important antioxidant molecule present in cells and protects against Cd
carcinogenicity both through its antioxidant activity and potentially through chelation by its
cysteinyl sulfhydryl group. As we shall see, exposure of cells and animals to Cd results in the
oxidative stress; and many of the reactive oxygen species (ROS) that are generated following
exposure of cells to Cd are detoxified either by the action of GSH or enzymes, such as GSH
peroxidase and GSH reductase, that are involved in the GSH redox cycle. You should have some
concept of the relation of ROS to chemical carcinogenesis from the section of the course that we
just concluded.
Heat shock proteins are a class of stress response protein induced in response to exposure to
physical and chemical insults and the induction of these proteins is considered as an adaptation
of the cells allowing them to perform functions essential for survival under conditions of stress.
It has been hypothesized that protein denaturation or any other type of protein damage caused by
Cd serves as the stimulus for induction of the genes encoding heat shock proteins.
Transcription factors
Some of the genes that are induced by the exposure of cells to Cd encode transcription factors
with resulting transcriptional deregulation of their target genes. The transcriptional genes include
the IEGs we have already mentioned, c-fos, c-jun, and c-myc but a host of other transcription
factors are also activated, including the metal regulatory transcription factor 1 (MTF1), which
binds to the MRE (metal response element) of promoters; upstream stimulator factor (USF),
which binds in the promoter region of the MT-1 gene; nuclear factor κB (NFκB) and NF-E2related factor 2 (NRF2). NF-κB regulates many physiological processes, including apoptosis, cell
adhesion, and cell proliferation. NF-κB can both induce and repress gene expression by binding
to particular DNA sequences, known as κB elements in promoters and enhancers. NF-κB is
inactivated and retained in the cytoplasm by complexation with a family of inhibitory proteins
called inhibitors of NF-κB (IκB). Activation of NF-κB involves the phosphorylation of IκB by
the IκB kinase (IKK) complex, which results in IκB degradation, allowing translocation of NFκB to the nucleus to transactivate (or repress) target genes. Nrf2 is a key transcription factor in
the transcriptional regulation of antioxidant response element-dependent (ARE-dependent) gene
expression in response to oxidative stress. Important Nrf2-regulated genes include heme
oxygenase-1 and NAD (P)H:quinone oxidoreductase-1. Heme oxygenase-1 degrades the heme in
hemoglobin to biliverdin and bilirubin, which are potent antioxidants and appear to be involved
in a manifold of additional protective physiological situations. NADPH:quinone oxidoreductase
(NQO1), is a dimeric flavoprotein that catalyzes the two-electron reduction of quinones to
hydroquinones. This reduction by 2 electrons prevents the one-electron reduction of quinones by
cytochrome P450 reductase and other flavoproteins that would otherwise result in redox cycling
of the quinones with generation of superoxide (O2−/●).
Translation factors
Translation factors are involved in the regulation of initiation, elongation and termination of
peptide chain synthesis – an area which we didn’t cover in our very cursory description of
protein synthesis. Overexpression of several of the translation factors has been identified in
cancer cell lines and tumor samples. In cells transformed by Cd, overexpression of translation
initiation factor 3 and translation elongation factor 1δ has been demonstrated.
Miscellaneous genes
“Miscellaneous genes” represents the large number of additional genes differentially expressed
in response to Cd exposure that have been identified by the use of microarrays. However, their
relation to Cd carcinogenesis remains to be demonstrated, which explains the “miscellaneous
category”. The following slide shows some of the genes activated.
[slide 4 summary of some activated genes]
Cadmium and the inhibition of DNA damage repair
We have discussed the fact a number of the responses to Cd involve defense against oxidative
stress. While genotoxicity induced by Cd is not a direct effect of the metal through the
generation of oxy radicals, exposure of cells to Cd results in the generation of 8-oxodG — a
reliable marker for oxidative DNA damage. What is the explanation? The explanation probably
lies in the observation that Cd exhibits the potential to inhibit DNA damage repair, and this has
been proposed as a major mechanism underlying the carcinogenic potential of Cd. The potential
of Cd to inhibit DNA damage repair has been demonstrated by several studies. Exposure of
alveolar epithelial cells to Cd significantly reduced the activity of formamido pyrimidine DNA
glycosylase, an enzyme involved in the recognition and removal of FapyG and FapyA as well as
other oxidative DNA damage such as 8-oxoG and 8-oxoA. The Fapy glycosylase contains a Znfinger binding motif and the Cd-induced inhibition of DNA damage repair is likely a result of
substitution for Zn. The zinc finger proteins substituted with Cd do not perform their functions as
efficiently as DNA damage repair enzymes with the appropriate Zn coordination. An important
implication of DNA repair inhibition by Cd is that Cd may also enhance the malignant
transformation of cells induced by other genotoxic chemicals.
.
Cadmium and apoptosis
We have learned how apoptosis plays an essential role in the elimination of damaged cells which
are potentially tumorigenic and we have by implication indicated that to survive cancer cells
develop mechanisms to avoid apoptosis. The potential involvement of apoptotic inhibition as a
mechanism for Cd carcinogenesis has been demonstrated by studies with normal human prostate
epithelial cells exposed to CdCl2. Two-thirds of the cells apoptosed - based on morphological
changes (e.g. appearance of fragmented DNA and other histological changes specific to
apoptosis) – but a third of the cells survived and showed resistance to Cd-induced apoptosis. So
it appears that in spite of its ability to induce apoptosis in cells, Cd may facilitate the selective
enrichment of a population of genetically damaged and apoptosis-resistant cells. A prominent
characteristic of the Cd-resistant population of cells was 2.5-fold greater MT content compared
to the normal, untreated cells. Gene expression profiling has provided insight into the molecular
mechanisms responsible for resistance to apoptosis in the selection of this population. Results of
cDNA microarray analysis of the gene expression profile demonstrated the down-regulation of
genes encoding several members of the caspase family of apoptotic proteases. The next slide
should refresh your memory about the role of caspases.
[Slide 5, repeat of receptor-mediated apoptosis pathway]
You recall that caspase-3 and caspase-9 were integral proteins involved in receptor-mediated
apoptotic pathways. Furthermore, the expression of bax, an important pro-apoptotic gene we
mentioned, was significantly reduced in the transformed cells compared with the control cells. At
the same time, the anti-apoptotic gene, bcl2, was significantly overexpressed in the transformed
cells compared to the controls.
The proposed role of Cd as an inhibitor of apoptosis is also cited with respect to its potential as a
co-carcinogen. Cd has been reported as an inhibitor of apoptosis induced by both metallic and
non-metallic toxic agents. The ability of Cd to block or inhibit apoptosis induced by hexavalent
chromium, known carcinogen, has been studied in Chinese hamster ovary (CHO K1-BH4) cells
treated with CdCl2 alone, hexavalent chromium alone or chromium plus CdCl2, and apoptosis
was determined 48-hour post-exposure. Exposure of the cells to chromium alone resulted in the
induction of apoptosis as evidenced from the appearance of DNA fragmentation and apoptotic
nuclei in the cells. However, Cd was able to block or inhibit chromium-induced apoptosis when
the cells were co-exposed to both metals. The inhibitory effect of Cd on chromium-induced
apoptosis is mediated through the inhibition of caspase 3 activity. Similar inhibitory effects of
CdCl2 have been reported on apoptosis induced by the antineoplastic drugs cisplatin and
etoposide. Cd has also been shown to inhibit apoptosis induced by benzo(a)pyrene-7,8-diol
epoxide (BPDE) in mammalian cells treated with Cd and BPDE.
As already indicated in describing the treatment of the human prostate epithelial cells with Cd,
Cd can induce apoptosis as well as inhibiting apoptosis. Multiple mechanisms appear to be
involved in the Cd-induced apoptosis. Both caspase dependent and independent mechanisms
involving mitochondria have been reported. In the case of mouse macrophages, oxidative stress
has been demonstrated as the major mechanism responsible for apoptosis. The oxidative stress in
the macrophages affected apoptosis indirectly by modulating the cellular level of Ca2+ and the
activities of caspases and mitogen activated protein kinases (MAPKs) in the cells. The role of
oxidative stress was confirmed by inhibition of apoptosis in the presence of antioxidants, Nacetyl cysteine (NAC), glutathione, and catalase.
Induction of ROS
We have discussed the activation of antioxidant defenses in response to Cd exposure, including
specific genes involved and the fact that oxidative damage is observed in Cd-exposed cells.
There is definitive evidence for the increased presence of free radicals in intact animals
following acute Cd overload. Superoxide anion, hydrogen peroxide, and hydroxyl radicals in
vivo have been detected by spectroscopic techniques. The expression of ROS-related genes in
response to Cd-overload just described gives strong biological support to the importance of
oxidative stress. In microarray analyses, in addition to the antioxidant gene products described
above, oxidative stress protein A170, heat-shock proteins, and oxidative DNA damage
responsive GADD45, GADD153, is increased. Conversely, the expression of genes encoding
metabolism is generally suppressed, which is interpreted as an attempt to switch the cellular
energy to overcome oxidative stress. (Metabolism can be a source of ROS.) It has been
suggested that the mechanisms of acute Cd toxicity involve the depletion of glutathione and
protein-bound sulfhydryl groups, resulting in enhanced production of ROS such as superoxide
ion, hydrogen peroxide, and hydroxyl radicals. The next slide illustrates proposed role of acute
Cd exposure in effects of ROS.
[slide 6, summarizing the effect of Cd on ROS]
In support of this hypothesis, the Fe-chelator Desferal can abolish the Cd-generated spintrapping of radical adducts in the bile, clearly indicating the involvement of endogenous irondependent hydroxyl radical generation (Fenton reaction) as a mechanism of Cd induced
oxidative stress. This observation is consistent with the fact that, in addition to background levels
of Fe, displacement of Fe from iron storage protein ferritin appears to be involved. Increased
ROS effects are amplified by lipid peroxidation, and results in DNA damage via the generation
of the α,β-unsaturated aldehydes. So overall, it is generally agreed upon that oxidative stress
plays important roles in acute Cd poisoning. However, following long-term Cd exposure at
environmentally-relevant low levels, direct evidence for oxidative stress is less well defined and
there are conflicting reports about its role. Alterations in ROS-related gene expression during
chronic exposures are less significant compared to acute Cd poisoning. This is probably due to
induced adaptation mechanisms such as increased levels of metallothionein and glutathione with
chronic Cd exposures, which would be expected to reduce Cd-induced oxidative stress. This is
supported by the observation that in chronic Cd-transformed cells, attenuated levels of ROS
signals are detected with spectroscopic probes. It may be that ROS are generated following acute
Cd overload and play important roles in tissue damage, and adaptation to chronic Cd exposure
reduces ROS production, but acquired Cd tolerance along with aberrant gene expression
probably plays important roles in chronic Cd toxicity and carcinogenesis.
With regard to aberrant gene expression, an epigenetic mechanism for Cd carcinogenesis may
also play a role through the ability of Cd to affect DNA methylation status. Cd can induce DNA
hypomethylation initially following acute exposure (transcription ↑), and subsequently induce
DNA hypermethylation (transcription ↓) following the long-term exposure at low
doses/concentrations. Cd-induced DNA hypermethylation in Cd-transformed prostate epithelial
cells is associated with increases in DNA methyltransferases activity and decreases in oxidative
stress and the redox-sensitive signal transduction pathways such as the JNK apoptotic pathway,
resulting in apoptotic resistance. (Remember the slide on receptor mediated apoptotic pathways
which included the non-caspase JNK pathway. The implication of increased methyltransferase
activity is that the transcription of the JNK pathway proteins is shut down.) Cd-induced DNA
hypermethylation was also shown to decrease the expression of tumor suppressor genes
(including p16), which could be an additional factor supporting Cd-induced malignant
transformation in human prostate cells.
Recently a theory to explain cadmium carcinogenesis has been formulated involving a protein
we have already discussed as an oncogene product, β-catenin
[Slide 7, repeat β-catenin]
The story originates with a protein called E-cadherin, which is a transmembrane, Ca2+-binding
glycoprotein, that plays a role in Ca2+-dependent cell–cell adhesion and is localized at the
adhesion belts of the adhering junctional complexes.
[Slide 8, showing role of E-cadherin]
E-cadherin has an intracellular domain that is linked to the actin cytoskeleton through catenins,
and an extracellular domain that contains the Ca2+-binding sites, in addition to the adhesion
domain of the molecule. Normally, Ca2+-binding to E-cadherin causes the protein to rigidify, and
it constrains the position of the adhesion sites in the molecule to those suitable for the formation
of a uniform cell–cell adhesion lattice. Cadmium was found to bind to a polypeptide which
corresponds to one of the extracellular Ca2+-binding regions of E-cadherin, changing its
conformation. The disruption of E-cadherin-mediated cell-adhesion triggers β-catenin-mediated
gene activation and this may represent early steps in the initiation phase of cancer. Remember
that in the cytosol, β-catenin can be phosphorylated and degraded or translocated to the nucleus
where it binds to transcription factors and alters the expression of several genes including c-myc
and c-jun. Because calcium activates E-cadherin and suppresses β-catenin, the displacement of
calcium from E-cadherin by cadmium possibly contributes to abnormal differentiation and
malignant progression. Supporting this pathway is the observation that after cadmium exposure
of renal tubule epithelial cells there is a loss of E-cadherin from the cell borders, the appearance
of gaps between cells, a decrease in the amount of β-catenin in the cell border and an increase in
its amount in the nuclei.
Begin 04/22/10
With regard to aberrant gene expression, an epigenetic mechanism for Cd carcinogenesis may
also play a role through the ability of Cd to affect DNA methylation status. Cd can induce DNA
hypomethylation initially following acute exposure (transcription ↑), and subsequently induce
DNA hypermethylation (transcription ↓) following the long-term exposure at low
doses/concentrations. Cd-induced DNA hypermethylation in Cd-transformed prostate epithelial
cells is associated with increases in DNA methyltransferases activity and decreases in oxidative
stress and the redox-sensitive signal transduction pathways such as the JNK apoptotic pathway,
resulting in apoptotic resistance. (Remember the slide on receptor mediated apoptotic pathways
which included the non-caspase JNK pathway.) The implication of increased methyltransferase
activity is that the transcription of the JNK pathway proteins is down-regulated.) Cd-induced
DNA hypermethylation was also shown to decrease the expression of tumor suppressor genes
(including p16), which could be an additional factor supporting Cd-induced malignant
transformation in human prostate cells. The mechanism by which methylation status is altered
has not been worked out.
Recently a theory to explain cadmium carcinogenesis has been formulated involving a protein
we have already discussed as an oncogene product, β-catenin
[repeat β-catenin slide]
The story originates with a protein called E-cadherin (= Ca adhesion), which is a
transmembrane, Ca2+-binding glycoprotein, that plays a role in Ca2+-dependent cell–cell
adhesion and is localized at the adhesion belts of the junctional complexes.
[slide showing role of E-cadherin]
E-cadherin has an intracellular domain that is linked to the actin cytoskeleton through catenins,
and an extracellular domain that contains the Ca2+-binding sites, in addition to the adhesion
domain of the molecule. Normally, Ca2+-binding to E-cadherin causes the protein to rigidify, and
it constrains the position of the adhesion sites in the molecule to sites appropriate for the
formation of a uniform cell–cell adhesion lattice. Cadmium has been found to bind to a Ca2+binding region of E-cadherin, changing its conformation. The disruption of E-cadherin-mediated
cell-adhesion triggers β-catenin-mediated gene activation and this may represent early steps in
the initiation phase of cancer. Remember that in the cytosol, β-catenin can be phosphorylated and
degraded or the unphosphorylated protein is translocated to the nucleus where it binds to
transcription factors and alters the expression of several genes including c-myc and c-jun as well
as genes involved in cell development. The excess β-catenin released in an unphosphorylated
form following E-cadherin deactivation translocates to the nucleus and possibly contributes to
abnormal differentiation and malignant progression. Supporting this pathway is the observation
that after cadmium exposure of renal tubule epithelial cells there is a loss of E-cadherin from the
cell borders, the appearance of gaps between cells, a decrease in the amount of β-catenin in the
cell border and an increase in its level in the nuclei.
CHROMIUM
Cr(VI) Metabolism. Cr is taken up by cells only in its hexavalent form. At neutral pH, Cr(VI)
exists as a mixture of chromate (CrO42–) and hydrochromate (HCrO4–) anions in the approximate
ratio of 3:1. Chromates are isostructural with physiological sulfate and phosphate ions, and this
molecular mimicry is why Cr(VI) readily enters cells through the sulfate channels. Most
chromates are sparingly soluble, and therefore absorption occurs under circumstances where
locally high concentrations can form. This is favorable in the lung, and absorption and
toxic/carcinogenic activity is pretty much confined to the lung. Human and other mammalian
cells are capable of massive accumulation of Cr(VI), with intracellular levels 10–20 times above
extracellular levels within 3 h. Cr(VI) is completely unreactive toward DNA under physiological
pH and temperature. In the biological systems, however, Cr(VI) undergoes a series of reduction
reactions ultimately yielding thermodynamically stable Cr(III).
[Slide 9, Cr uptake and metabolism]
When this occurs extracellularly, reduction acts as a detoxification process because membranes
are only poorly permeable to Cr(III). Inside the cell, Cr(VI) reduction is the activation event that
is responsible for the generation of genotoxic damage and other forms of toxicity. Cr(VI)
metabolism in mammalian cells consists of direct electron transfer from ascorbate and
nonprotein thiols, such as glutathione and cysteine. Ascorbate is the dominant biological reducer
of Cr(VI), accounting for about 90% of its metabolism in cells in vivo. Cultured cells typically
contain < 50–60 μM ascorbate and rely on thiols for Cr(VI) reduction, so unless cellular
ascorbate levels are restored to normal by supplementation, cell cultures are not a true
physiological model of Cr(VI) metabolism. While the end-product of Cr(VI) metabolism is
always Cr(III), the reduction process can generate variable amounts of transient Cr(V), Cr(IV),
and organic radicals depending on the reducing agent and the ratio of reactants. The significance
of this observation is that under physiological conditions, that is high ascorbate concentrations,
the high-valent forms Cr(IV) and Cr(V) are not a significant actors. This observation is important
because considerable effort has been devoted to investigating the role of Cr(IV) and Cr(V) in the
genotoxic and mutagenic capability of Cr, particularly with regard to oxidative stress. Thus a
large segment of literature devoted to the activity of high-valent transients in the reduction
cascade probably does not have physiological relevance. The final product of Cr(VI)
metabolism, Cr(III), forms stable coordination complexes with nucleic acids and proteins.
Studies in Cr(VI)-treated cells and in vitro reduction reactions under physiologically realistic
conditions show the formation of several types of DNA damage, including strand breaks and
various Cr-DNA adducts.
Cr-DNA Adducts. Low molecular weight Cr-DNA adducts are the most abundant form of
Cr(VI)-induced genetic lesions in mammalian cells, and they were found to be responsible for all
mutagenic damage generated during Cr(VI) reduction with both cysteine and ascorbate. Some
structures are on the next slide.
[Slide 10: structures of Cr(III)-DNA adducts]
The 50–75% of adducts in vitro are binary Cr-DNA complexes and these are only weakly
mutagenic. Their existence in vivo is debated because ternary Cr-DNA adducts can be disrupted
during DNA isolation to produce binary adducts which of course complicates the assessment of
the actual levels of binary adducts. The predominant forms of Cr-DNA complexes in cells are
ternary adducts (cross-links), in which the Cr(III) atom bridges DNA with small cellular
molecules (L-Cr-DNA). Four major forms of ternary adducts are glutathione-Cr-DNA, cysteineCr-DNA, histidine-Cr-DNA, and ascorbate-Cr-DNA complexes. All ternary adducts are much
more mutagenic than binary adducts, and ascorbate-Cr-DNA crosslinks were the most potent
promutagenic of the Cr-DNA modifications. Ternary adducts are formed through an attack of
DNA by ligand-Cr(III) complexes. The primary site of attachment for all Cr(III) adducts is the
phosphate group, but induction of G:C-targeted mutagenic events by Cr-DNA modifications has
also led to the suggestion that the mutagenic forms of adducts are probably Cr(III) microchelates
involving a phosphate group and the N7 position of G (slide). The adducts are substrates for
nucleotide excision repair (NER) in human and hamster cells, supported by the persistence of
total adducts and increased toxicity and mutagenicity of Cr-DNA damage in NER-deficient cells.
A recent assignment of Cr-dG binding to 5'-NGG-3' sequences, which was based on the mapping
of DNA nicks made by bacterial UvrABC mapping in Cr-adducted DNA, is consistent with this
sequence specificity of Cr-adduct mutagenesis.
DNA–Protein and DNA Interstrand Cross-Links. The formation of DNA–protein cross-links
(DPC) by Cr(VI) has been observed both in vivo and in vitro. Sensitive assays are available to
measure DPCs, and the overall yield of DPC in cells is small, estimated as less than 1% of total
Cr-DNA adducts. Because of the molecular volume of octahedral Cr(III) and the attached
protein, the lesions are extremely bulky and would be expected to block replication and
transcription, but at this time, the biological significance and repair of Cr-induced DPCs is
unknown. Interstrand DNA cross-links are an area of controversy. The conditions under which
interstrand cross-links have been identified involve Cr concentrations that are unrealistically
high, and result in the formation of Cr oligomers which would not exist in cells. At non-lethal
concentrations of Cr, interstrand cross-links are not expected to be important on the basis of the
severe steric restrictions for the intercalation of monomeric octahedral Cr(III) complexes and the
lack of Cr(VI) hypersensitivity in crosslink repair-deficient [ERCC4(XPF)-null] CHO cells
provides a strong argument that interstrand DNA cross-linking does not contribute to Cr
genotoxicity.
DNA Breaks. The presence of single-strand breaks (SSB) in chromate-treated cells in culture
and in animal tissues has been reported in several studies that used standard detection assays for
the quantitation of these DNA lesions. The assays all involved treatment of DNA under strongly
alkaline conditions raising a major concern as to whether the data measured genuine SSB or
breaks that were artifacts of the alkaline assay conditions. As in the case of other phosphate
triesters, Cr-DNA phosphate adducts make the phosphate backbone linkages unstable under
alkaline conditions, causing breaks. Another complication in the detection of SSB attributed to
oxidative damage is the presence of strand breaks resulting from the rapid excision of Cr-DNA
adducts by NER, which generates 50,000 excision events/min in human cells following exposure
even to non-toxic Cr(VI) concentrations (2–5 μM). The occurrence of Cr-mediated SSBs is
supported only at high, toxic doses of Cr by comparing Cr(VI) toxicity in SSB-repair deficient
(XRCC1–/–) and proficient (XRCC1+) EM9-CHO cells. The production of SSB was inhibited by
the addition of catalase and iron chelators implicating involvement of Fenton chemistry and
H2O2. Although Cr(V) is known be involved in oxy radical generation by Fenton chemistry, the
levels of Cr(V) in cells with physiological levels of ascorbate are negligible, so in the absence of
extraneous Fe(II), hydroxyl radical should not be a factor. In fact, careful sample preparation to
exclude Fe greatly reduces the SSB observed. The presence of H2O2 needs to be explained, but
could be a result of mitochondrial damage or elevated activity of NADPH oxidases.
Recently, evidence for the formation of DSB in Cr(VI)-treated human cells has been obtained.
The DSB were produced via an indirect mechanism, which required the passage of cells through
S-phase and the participation of mismatch repair proteins. (We will come back to this in a few
minutes.) The next slide summarizes pathways leading to SSBs and DSBs.
[Slide 11, major pathways leading SSB and DSB]
DNA Base Damage. Administration of Cr(VI) to animals with different tissue levels of
ascorbate failed to induce the formation of 8-oxoG, which is the most widely used indicator of
the oxidative insult on DNA. However, there is a recent report that Cr(VI) yielded Sp in vivo in
E. coli treated with > 100 μM K2CrO7 (we cited the result in the unit on oxidative damage) and
also in vitro in dsDNA by reduction of K2CrO7 (at lower concentrations (3 – 50 μM) with
ascorbate. Since Sp is a further oxidation product of 8-oxoG, the 8-oxoG should have initially
been present. In the in vitro work, 8-oxoG was detected at a level ~5% of Sp and the rapid
conversion to Sp in vivo might explain why 8-oxoG was not detected. Sp was first characterized
in 2001, and even now, would not be routinely assayed, so it could have been overlooked in all
other studies – identification of Sp would imply 8-oxoG as a precursor. The generation of Sp in
vivo or in vitro with excess ascorbate would seem to be inconsistent with the suggestion that
oxidative stress should not be important under physiological relevant conditions, but an element
of uncertainty in vivo arises from the fact that the concentration of Cr(VI) used to treat the cells
was high and perhaps ROS originated from damage to the aerobic respiration chain. To settle this
point, determination of spiroiminodihydantoin and other advanced oxidation products of guanine
in ascorbate-complemented human cells is necessary.
Begin 04/27/10
Genomic Instability, Toxicity, and Cr(VI) Carcinogenesis. One feature characteristic of
Cr(VI)-associated cancers is the presence of microsatellite instability, which is indicative of a
defective mismatch repair (MMR). Microsatellite DNA consists of multiple repeats of short
sequences that are interspersed throughout the genome. In Cr-induced cancers, an elevated
mutation rate within microsatellites is associated with the loss of expression of MLH1, which
you might remember is the human homolog of mutL, one of the essential MMR proteins in E.
coli. MMR is a critical system used by cells to correct replication errors, and cells deficient in
MMR exhibit mutation rates 100-times higher than normal within genes, and at even greater
rates within the microsatellites. Thus, chromate-associated cancer cells express a phenotype
called a “mutator phenotype” because of the increasing propagation of mutations caused by the
loss of a major mutation avoidance system. Once MMR is inactivated, the rate of mutation in the
critical growth-controlling genes is greatly accelerated since the mutator phenotype maintains
high rates of random mutagenesis. An interesting consequence is that exposure to Cr(VI) for
additional mutagenic events is no longer required for the cell to progress to a transformed state.
The question arises as to how Cr(VI) selectively leads to the appearance of this specific form of
microsatellite instability, which is uncommon for other lung carcinogens. One hint is that (1) the
absence of MMR eliminated the ability of Cr-DNA adducts to inhibit cellular replication of Crmodified vectors and (2) concurrently elimination of MMR reduced the induction of DSB in
Cr(VI)-treated cells: that is, no MMR = no DSB. Thus, MMR appears to be responsible for an
aberrant repair process in which DSBs are generated. The Cr-resistant phenotype can be induced
by the loss of any of the main MMR proteins (in addition MLH1 that includes MSH2, MSH6 or
PMS2), indicating that the entire MMR complex is required for the processing of Cr-DNA
adducts into highly toxic DSB. The damage-promoting effects of MMR extended over a range of
Cr(VI) concentrations from very low, nontoxic (<1 μM within drinking water standards) to
highly toxic doses (>90% clonogenic lethality). The potentiating effects of MMR were most
strongly pronounced in cells supplemented with physiological levels of ascorbate. MMRpromoted DSB were preferentially found in the G2 phase of the cell cycle, irrespective of dose,
postexposure time, and type of cell. The G2 specificity of DSB production is presumed to result
from the requirement for Cr-damaged DNA to undergo replication, i.e., pass through the S-
phase, in order for the MMR system to activate aberrant processing. These observations have led
to a proposed model for transformation in which highly mutagenic adducts such as the
ascorbate–Cr-DNA cross-links described in the last lecture induce mismatches during the
replication of damaged DNA and the compound lesions (consisting of mismatches at the site of
Cr adducts) lead to abnormal MMR. In this scenario, adducts both induce mutations and also
promote larger chromosomal abnormalities including DSB. The DSB are handled through an
error-prone system known as nonhomologous end-joining. We did not cover nonhomologous end
joining, but suffice it to say for now that NHEJ is a process involving resection of the ends of
dsDNA at double strand breaks, and thus results in deletions and translocations (the exchange of
chromosomal material). The error-prone repair can eventually select for a MMR-deficient
population, since these cells have a growth advantage . The observed tolerance of Cr(VI) by
MMR-deficient cells (meaning this doesn’t happen in the absence of MMR) and the absence of
MMR in chromate-induced lung cancers led to the formulation of the selection model for Cr(VI)
carcinogenesis shown in the next slide (MSI+ = microsatellite instability).
[slide, model for MMR-induced cell transformation]
This model postulates that chronic exposure to toxic doses of Cr(VI) results in the selective
growth of resistant cells that lack MMR, although, as we have just said, MMR is important in
initiating the pathway. Once a population of MMR-deficient cells emerges, the subsequent
exposure to Cr(VI) may no longer be necessary for the generation of additional mutations needed
for the further progression of initiated cells because MMR-null cells have very high rates of
spontaneous mutagenesis.
Direct involvement of Cr(VI) in cell signaling. The involvement of Cr in cell signaling is much
less well defined than for Cd. Much of the gene expression observed with Cr-exposure may be
due to damage response or epigenetic effects rather than direct action of Cr. However a recently
published study does provide at least one concrete example of activity directly induced by Cr.
Exposing airway epithelial cells to Cr(VI) increases DNA binding and promoter transactivation
by members of a protein family called “signal transducer and activator of transcription” (STAT)
including STAT1 which suppresses expression of the protein, “vascular endothelial cell growth
factor A” (VEGFA). VEGFA is an important mediator of lung injury and repair. The STATs are
activated through the phosphorylation of a conserved tyrosine residue in the C-terminus by a
kinase Fyn, which is a Cr(VI)-stimulated src family kinase (SFK) upstream of STAT1 in the
activation pathway. The role of Fyn was confirmed by demonstrating that Cr(VI) failed to
activate STAT1 in cells that lacked Fyn and restoring Fyn, provided a fully functional
STAT1response to Cr(VI). Cr(VI) stimulates Fyn kinase activity through a direct effect on the
enzyme, demonstrated by the fact that substrate phosphorylation was increased by adding Cr(VI)
to a solution containing only affinity purified enzyme, substrate, and ATP. The effect of Cr(VI)
was not inhibited in the presence of catalase but was prevented by the addition of a Cr chelator,
N-acetyl Cys to the reaction mixture. This suggests that Cr(VI) reacts with a thiol in the kinase.
Cr(VI) causes similar activation of recombinant Src under the same reaction conditions. The
thiol dependence for the activation of purified Fyn or Src is consistent with structural studies that
identified a metal-responsive motif in a carboxyl regulatory domain of the SFK enzymes that
contains two critical cysteines separated by 10 amino acids. Conformational change in this motif
during activation is recognized to increase substrate affinity. Whether Cr(VI) binds these
cysteines or binds to other amino acids in the regulatory domain in intact cells or in vivo remains
to be determined.
Cr(VI) as a Cocarcinogen. The possibility of a role for Cr(VI) as a cocarcinogen had been
debated for a long time, but only within the last few years has chromate cocarcinogenesis
actually been demonstrated in animal studies and insights into mechanism have been obtained.
Experimental data definitively show that Cr(VI) can act as a potent cocarcinogen for UVinduced skin tumors. In these studies, the presence of Cr(VI) in drinking water caused dosedependent increases in the frequency of skin tumors in UV-irradiated hairless mice. Cr(VI) alone
produced no tumors, indicating that it acted a strong enhancer of the UV-initiated tumorigenesis.
Supplementation of the animals’ diets with the antioxidants vitamin E or selenomethionine
(methionine with Se substituted for S) had no effect on Cr(VI)-mediated enhancement of skin
carcinogenesis, suggesting that cocarcinogenic effects were not oxidant-mediated. Since the
inability to repair UV-induced DNA damage would be a logical cause of the observed skin
cancer, a likely target of Cr-UV synergism could be interference of Cr(VI) with NER of UVinduced pyrimidine dimers. We have already discussed the observation that Cr-DNA adducts,
which occur at very high levels even with non toxic Cr(VI) intake, are substrates for human NER
and are rapidly removed. Thus, the presence of Cr-DNA adducts in UV-irradiated keratinocytes
can be expected to compete for NER machinery while mutagenic UV-DNA damage, which is
removed at a slower rate, will persist. For example, a 3-h treatment of primary human fetal lung
cells (IMR90) with 2 μM Cr(VI), a dose in water that meets the current federal standard for Cr in
drinking water, caused 107 Cr-DNA adducts/cell. On the basis of a t1/2 = 8.2 h in the fetal lung
cells, total co-opting of cellular NER would still leave about 106 Cr lesions/genome 24 h
postexposure. Hence, the comutagenicity of Cr(VI) and UV can plausibly be explained by
competition for NER factors. Tobacco smoke and Cr(VI) exposure represent another potential
case for synergistic tumorigenesis. DNA adducts formed by polycyclic aromatic hydrocarbons,
which are one of the main groups of tobacco-derived mutagens, are repaired by NER. A study
found preexposure of CHO cells to Cr(VI) led to a significantly slower repair of BPDE-DNA
adducts, which was accompanied by increased cytotoxicity and mutagenesis of BPDE.
Coexposure to Cr and tobacco smoke produced no synergism repair-deficient cells, which clearly
points to NER as the key target of enhancement of BPDE genotoxicity by Cr(VI). Interestingly,
Cr(VI) appears to cause a selective increase in the number of BPDE adducts at the mutational
hotspots of p53 in smoking-induced lung cancer: codons 248, 273, and 282. These sites are likely
to be repair coldspots, which would make them particularly sensitive to decreased NER because
of the competition with Cr-DNA adducts.