Air Quality Criteria for Lead Volume II of II - (NEPIS)(EPA) - US ...

AX5-133
Table AX5-8.3 (cont’d). Bone Cell Cultures Utilized to Test Effects of Lead
Compound
Dose/Concentration
Duration Exposure
Route Species Effects Blood Level Reference
Unidentified Pb 2+
Various incubation
times
Not applicable
Unidentified Pb 2+
Various incubation
times
Not applicable
Pb acetate
10 µM
2 h
In medium
Pb acetate
5 or 25 µM
Up to 24 h
In medium
Pb acetate
5 or 25 µM
20 h
In medium
Bovine
(Bovinederived
osteocalcin)
Bovine
(Bovinederived
osteocalcin)
Rat
Osteosarcoma
Cells
(ROS 17/2.8)
Rat
Osteosarcoma
Cells
(ROS 17/2.8)
Rat
Osteosarcoma
Cells
(ROS 17/2.8)
Binding studies of Ca 2+ to osteocalcin suggested a single binding site with a
dissociation constant (Kd) of 7 ± 2 µM for Ca-osteocalcin. Competitive displacement
experiments by addition of Pb 2+ indicated the Kd for Pb-osteocalcin is 1.6 ± 0.42 nM,
~3 orders of magnitude higher.
Circular dichroism indicated Pb 2+ binding induced a structural change in osteocalcin
similar to that found in Ca 2+ binding, but at 2 orders of magnitude lower concentration.
Pb 2+ has 4 orders of magnitude tighter binding to osteocalcin (Kd = 0.085 µM) than
Ca 2+ (Kd = 1.25 mM). Hydroxyapatite binding assays showed similar increased
adsorption of Pb 2+ and Ca 2+ to hydroxyapatite, but Pb 2+ adsorption occurred at a
concentration 2–3 orders lower than Ca 2+ .
Pb 2+ treatment reduced the unidirectional rate of ATP synthesis (P i to ATP) by a factor
of 6 or more (∆M/M o: Control = 0.18 ± 0.04, Pb 2+ < 0.03). Intracellular free Mg 2+
concentration decreased 21% after 2 h of 10 µM Pb 2+ treatment (0.29 ± 0.02 mM prior
to Pb 2+ treatment and 0.23 ± 0.02 mM after 2 h of Pb 2+ treatment, p < 0.05).
5 µM Pb 2+ significantly altered effect of EGF on intracellular calcium metabolism.
In cells treated with 5 µM Pb 2+ and 50 ng/mL EGF, there was a 50% increase in total
cell calcium over cells treated with 50 ng/mL EGF alone.
Treatment with 400 ng/mL culture medium for 1 h or with 25 µM Pb 2+ for 20 h
increased total cell calcium:
Treatment Cell Calcium
Control 7.56 ± 1.05 nmol/mg protein
PTH (400 ng/mL) 23.28 ± 1.40* nmol/mg protein
Pb (25 µM) 11.37 ± 0.57* nmol/mg protein
PTH + Pb 37.88 ± 4.21* nmol/mg protein
* p # 0.05 from control
Not applicable Dowd et al. (1994)
Not applicable Dowd et al. (2001)
Not applicable Dowd et al. (1990)
Not applicable Long and Rosen
(1992)
Not applicable Long et al. (1992)